CN113474352A - Novel phosphoramidites - Google Patents

Abstract

The invention relates to a compound of formula (II), wherein X, Y, R⁵、R^x、R^yAnd Nu is as defined in the description and claims. The compounds of formula (II) are useful for the manufacture of a medicament.

Description

Novel phosphoramidites

The invention relates in particular to a single-stranded antisense gapmer oligonucleotide comprising at least one dinucleoside of formula (I)

Wherein (A)¹) And (A)²) One is a sugar modified nucleoside, the other is a sugar modified nucleoside or a DNA nucleoside, and a is oxygen or sulfur, or a pharmaceutically acceptable salt thereof.

The invention also relates in particular to a novel phosphoramidite which can be used for the preparation of the antisense gapmer oligonucleotides according to the invention.

In recent years, significant progress has been made in the synthesis of oligonucleotides as therapeutics, resulting in a wide range of clinically validated combinations of molecules (including RNase H activated notch mers, splice switching oligonucleotides, microRNA inhibitors, siRNAs or aptamers) that act through different mechanisms (S.T. Crooke, Antisense drug technology: principles, strategies, and applications, 2 nd edition, Boca Raton, FL: CRC Press, 2008). Natural oligonucleotides are inherently unstable to nuclear degradation in biological systems. Furthermore, they exhibit very unfavorable pharmacokinetic behavior. To ameliorate these disadvantages, various chemical modifications have been studied in recent decades. One modification that is probably the most successful is the introduction of phosphorothioate linkages in which one of the non-bridging phospho-oxygen atoms is replaced by a sulfur atom (F. Eckstein, Antisense and Nucleic Acid Drug Development 2009,10, 117-121). Such phosphorothioate oligodeoxynucleotides exhibit increased protein binding and significantly higher stability to nucleolytic degradation compared to the unmodified phosphodiester analogues, and therefore have significantly higher half-lives in plasma, tissue and cells. These key features contribute to the development of first generation oligonucleotide therapies and pave the way for further improvements by later generations of modifications such as Locked Nucleic Acids (LNAs).

It has surprisingly been found that the single-stranded antisense oligonucleotides according to the invention are well tolerated. They are at least as effective in vitro as reference oligonucleotides comprising phosphorothioate internucleoside linkages alone and are more effective in vivo than reference oligonucleotides comprising phosphorothioate internucleoside linkages alone. It is also surprising that the single stranded antisense oligonucleotides according to the invention are particularly effective in cardiac cell lines (in vitro) and cardiac tissue (in vivo).

FIG. 1 shows a dose response curve of oligonucleotides according to the invention targeting MALAT1 mRNA in the human HeLa cell line.

Figure 2 shows a dose response curve of oligonucleotides according to the invention targeting MALAT1 mRNA in the human a549 cell line.

FIG. 3 shows a dose response curve for an oligonucleotide according to the invention targeting HIF1A mRNA in the human HeLa cell line.

FIG. 4 shows a dose response curve for an oligonucleotide according to the invention targeting HIF1A mRNA in the human A549 cell line.

Fig. 5 shows a dose response curve of oligonucleotides according to the invention targeting ApoB mRNA in mouse primary hepatocytes.

FIG. 6 shows the amount of Malat1 mRNA levels in hearts of animals treated with oligonucleotides according to the invention.

In the present specification, the term "alkyl" alone or in combination denotes a straight or branched alkyl group having 1 to 8 carbon atoms, particularly a straight or branched alkyl group having 1 to 6 carbon atoms, and more particularly a straight or branched alkyl group having 1 to 4 carbon atoms. Straight and branched C₁-C₈Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, the isomeric pentyl, the isomeric hexyl, the isomeric heptyl and the isomeric octyl groups, in particular methyl, ethyl, propyl, butyl and pentyl groups. Specific examples of alkyl groups are methyl, ethyl and propyl.

The term "cycloalkyl" alone or in combination denotes a cycloalkyl ring having 3 to 8 carbon atoms, and in particular a cycloalkyl ring having 3 to 6 carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, more particularly cyclopropyl and cyclobutyl. A specific example of "cycloalkyl" is cyclopropyl.

The term "alkoxy", alone or in combination, denotes a group of the formula alkyl-O-, wherein the term "alkyl" has the previously given meaning, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, secondary butoxy and tertiary butoxy. Particular "alkoxy" groups are methoxy and ethoxy. Methoxyethoxy is a specific example of "alkoxyalkoxy".

The term "oxy" alone or in combination denotes an-O-group.

The term "alkenyl", alone or in combination, denotes a straight-chain or branched hydrocarbon residue comprising an olefinic bond and up to 8, preferably up to 6, particularly preferably up to 4 carbon atoms. Examples of alkenyl are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl and isobutenyl.

The term "alkynyl", alone or in combination, denotes a straight-chain or branched hydrocarbon residue comprising a triple bond and up to 8, especially 2, carbon atoms.

The term "halogen" or "halo", alone or in combination, denotes fluorine, chlorine, bromine or iodine, especially fluorine, chlorine or bromine, more especially fluorine. The term "halo" in combination with another group means that the group is substituted with at least one halogen, particularly one to five halogens, particularly one to four halogens (i.e., one, two, three or four halogens).

The term "haloalkyl" alone or in combination denotes an alkyl group substituted by at least one halogen, in particular by one to five halogens, in particular one to three halogens. Examples of haloalkyl include monofluoro-, difluoro-or trifluoromethyl, -ethyl or-propyl, such as 3,3, 3-trifluoropropyl, 2-fluoroethyl, 2,2, 2-trifluoroethyl, fluoromethyl or trifluoromethyl. Fluoromethyl, difluoromethyl and trifluoromethyl are specific "haloalkyl" groups.

The term "halocycloalkyl" alone or in combination denotes a cycloalkyl group as defined above substituted by at least one halogen, in particular by one to five halogens, in particular one to three halogens. Specific examples of "halocycloalkyl" are halocyclopropyl, especially fluorocyclopropyl, difluorocyclopropyl and trifluorocyclopropyl.

The term "hydroxy" alone or in combination denotes an-OH group.

The term "mercapto" alone or in combination denotes the-SH group.

The term "carbonyl", alone or in combination, denotes a-c (o) -group.

The term "carboxyl" alone or in combination denotes a-COOH group.

The term "amino" alone or in combination denotes a primary amino group (-NH)₂) A secondary amino group (-NH-), or a tertiary amino group (-N-).

The term "alkylamino" alone or in combination denotes an amino group as defined above substituted by one or two alkyl groups as defined above.

The term "sulfonyl", alone or in combination, denotes-SO₂A group.

The term "sulfinyl" alone or in combination denotes a-SO-group.

The term "thio" alone or in combination denotes an-S-group.

The term "cyano", alone or in combination, denotes a-CN group.

The term "azido", alone or in combination, denotes-N₃A group.

The term "nitro", alone or in combination, denotes NO₂A group.

The term "formyl", alone or in combination, denotes the-C (O) H group.

The term "carbamoyl" alone or in combination denotes-C (O) NH₂A group.

The term "ureido" alone or in combination means-NH-C (O) -NH₂A group.

The term "aryl" alone or in combination, denotes a monovalent aromatic carbocyclic monocyclic or bicyclic ring system comprising 6 to 10 carbon ring atoms, said system being optionally substituted with 1 to 3 substituents independently selected from: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of aryl groups include phenyl and naphthyl, especially phenyl.

The term "heteroaryl" alone or in combination, denotes a monovalent aromatic heterocyclic monocyclic or bicyclic ring system of 5 to 12 ring atoms, said system comprising 1,2, 3 or 4 heteroatoms selected from N, O and S, the remaining ring atoms being carbon, optionally substituted with 1 to 3 substituents independently selected from: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of heteroaryl groups include pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepine, isoxazolyl, benzofuranyl, isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, carbazolyl, or acridinyl.

The term "heterocyclyl" alone or in combination, denotes a monovalent saturated or partially unsaturated monocyclic or bicyclic ring system of 4 to 12, especially 4 to 9, ring atoms, said system comprising 1,2, 3 or 4 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon, optionally substituted with 1 to 3 substituents independently selected from: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of monocyclic saturated heterocyclic groups are azetidinyl, pyrrolyl, tetrahydrofuryl, tetrahydrothienyl, pyrazolidinyl, imidazopyridinyl, oxazolidinyl, isoxazolidinyl, tetrahydrothiazolyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperidinyl or oxazepanyl. Examples of bicyclic saturated heterocycloalkyl are 8-aza-bicyclo [3.2.1] octyl, quinuclidinyl, 8-oxo-3-aza-bicyclo [3.2.1] octyl, 9-aza-bicyclo [3.3.1] nonyl, 3-oxo-9-aza-bicyclo [3.3.1] nonyl or 3-thio-9-aza-bicyclo [3.3.1] nonyl. Examples of partially unsaturated heterocycloalkyl groups are dihydrofuranyl, imidazolinyl, dihydrooxazolyl, tetrahydropyridinyl or dihydropyranyl.

The term "pharmaceutically acceptable salts" refers to those salts that retain the biological effects and properties of the free base or free acid, which are not biologically or otherwise undesirable. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. In addition, these salts can be prepared by addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, the following: salts of primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The oligonucleotides of the invention may also be present in zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the invention are the sodium, lithium, potassium and trialkylammonium salts.

The term "protecting group", alone or in combination, represents a group that is capable of selectively blocking a reactive site in a multifunctional compound, while allowing a chemical reaction to proceed selectively at another unprotected reactive site. The protecting group may be removed. Exemplary protecting groups are amino protecting groups, carboxyl protecting groups, or hydroxyl protecting groups.

"phosphate protecting group" is a protecting group for a phosphate group. Examples of phosphate protecting groups are 2-cyanoethyl and methyl. A specific example of a phosphate protecting group is 2-cyanoethyl.

"hydroxyl protecting group" is a protecting group for a hydroxyl group and also serves to protect a thiol group. Examples of hydroxy protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β -Methoxyethoxymethyl Ether (MEM), dimethoxytrityl (or bis- (4-methoxyphenyl) phenylmethyl) (DMT), trimethoxytrityl (or tris- (4-methoxyphenyl) phenylmethyl) (TMT), methoxymethyl ether (MOM), methoxytrityl [ (4-methoxyphenyl) diphenylmethyl (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), Tetrahydropyranyl (THP), Tetrahydrofuran (THF), trityl or trityl (Tr), silyl ethers (e.g. Trimethylsilylimidazole (TMS), tert-butyldimethylsilyl (TBDMS), Triisobutylsilyloxymethyl (TOM) and Triisopropylsilyl (TIPS) ether), Methyl ether and Ethoxyethyl Ether (EE). Specific examples of hydroxyl protecting groups are DMT and TMT, especially DMT.

"mercapto protecting group" is a protecting group for a mercapto group. Examples of thiol protecting groups are those of the "hydroxy protecting group".

If one of the starting materials or compounds of the invention contains one or more functional Groups which are unstable or reactive under the reaction conditions of one or more reaction steps, suitable protecting Groups can be introduced before applying the key steps of methods well known in the art (as described, for example, in "Protective Groups in Organic Chemistry", 3 rd edition, 1999, Wiley, New York, T.W. Greene and P.G.M.Wuts). Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature. Examples of protecting groups are tertiary butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonylamide (Fmoc), 2-trimethylsilylethylcarbonylamide (Teoc), benzyloxycarbonyl (Cbz) and p-methoxybenzyloxycarbonyl (Moz).

The compounds described herein may contain several asymmetric centers and may be in the form of pure enantiomers, mixtures of enantiomers such as racemates, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

Oligonucleotides

As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more covalently linked nucleosides as is commonly understood by a skilled artisan. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually prepared in the laboratory by solid phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the nucleobase portion of a covalently linked nucleotide or nucleoside or a modified sequence or order thereof. The oligonucleotides of the invention are artificial, chemically synthesized, and usually purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides or nucleotides.

Antisense oligonucleotides

The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly to a contiguous sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded and are therefore not sirnas or shrnas. Preferably, the antisense oligonucleotides of the invention are single stranded. It will be appreciated that single stranded oligonucleotides of the invention may form hairpin or intermolecular duplex structures (duplexes between two identical oligonucleotides) as long as the degree of internal or inter-self complementarity is less than 50% across the full length of the oligonucleotide.

Continuous nucleotide sequence

The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. The term is used herein interchangeably with the term "contiguous nucleobase sequence" and the term "oligonucleotide motif sequence". In some embodiments, all nucleotides of an oligonucleotide comprise a contiguous nucleotide sequence. In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence, such as a F-G-F' gapmer region, and may optionally comprise other nucleotides, such as a nucleotide linker region that can be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

Nucleotide, its preparation and use

Nucleotides are building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides. In practice, nucleotides, such as DNA and RNA nucleotides, include a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (which are not present in the nucleoside). Nucleosides and nucleotides can also be interchangeably referred to as "units" or "monomers".

Modified nucleosides

As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified compared to an equivalent DNA or RNA nucleoside by introducing one or more modifications of the sugar moiety or (nucleobase) moiety. In preferred embodiments, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer". Nucleosides having unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides that have modifications in the base region of a DNA or RNA nucleoside are still commonly referred to as DNA or RNA if Watson Crick base pairing is allowed.

Modified internucleoside linkages

As generally understood by the skilled artisan, the term "modified internucleoside linkage" is defined as a linkage other than a Phosphodiester (PO) linkage, which covalently couples two nucleosides together. Thus, the oligonucleotides of the invention may comprise modified internucleoside linkages. In some embodiments, the modified internucleoside linkage increases nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, internucleoside linkages include phosphate groups that result in phosphodiester linkages between adjacent nucleosides. The modified internucleoside linkages are particularly useful for stabilizing oligonucleotides for use in vivo, and for preventing nuclease cleavage at a DNA or RNA nucleoside region (e.g., within the notch region of a notch-mer oligonucleotide and in modified nucleoside regions, such as regions F and F') in oligonucleotides of the invention.

In one embodiment, the oligonucleotide comprises one or more internucleoside linkages modified with a native phosphodiester, e.g., one or more modified internucleoside linkages, which is more resistant to, e.g., nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay, such as Snake Venom Phosphodiesterase (SVPD), both of which are well known in the art. Internucleoside linkages capable of enhancing nuclease resistance of an oligonucleotide are known as nuclease-resistant internucleoside linkages. In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are modified, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, of the internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are nuclease-resistant internucleoside linkages. In some embodiments, all of the internucleoside linkages of the oligonucleotide or a contiguous nucleotide sequence thereof are nuclease-resistant internucleoside linkages. It will be appreciated that in some embodiments, the nucleoside linking the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be a phosphodiester.

A preferred modified internucleoside linkage for use in the oligonucleotide of the invention is phosphorothioate.

Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% of the internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate. In some embodiments, all internucleoside linkages of the oligonucleotide, or a contiguous nucleotide sequence thereof, other than the trithiophosphate internucleoside linkage, are phosphorothioates. In some embodiments, in addition to the trithiophosphate ester linkage, the oligonucleotides of the invention comprise phosphorothioate internucleoside linkages and at least one phosphodiester linkage, such as 2, 3, or 4 phosphodiester linkages. In a gapmer oligonucleotide, the phosphodiester bond (when present) is suitably not located between consecutive DNA nucleosides in the gap region G.

Nuclease-resistant linkages such as phosphorothioate linkages are particularly useful in regions of the oligonucleotide that are capable of recruiting nucleases when forming duplexes with the target nucleic acid, such as region G of the gapmer. However, phosphorothioate linkages may also be used in non-nuclease recruiting regions and/or affinity enhancing regions, such as regions F and F' of gapmers. In some embodiments, the gapmer oligonucleotide may comprise one or more phosphodiester linkages in region F or F 'or both regions F and F', wherein the internucleoside linkage in region G may be entirely phosphorothioate.

Advantageously, all internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide or all internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

It will be appreciated that antisense oligonucleotides may comprise other internucleoside linkages (other than phosphodiester and phosphorothioate) as disclosed in EP 2742135, for example alkylphosphonate/methylphosphonate internucleoside linkages which may be otherwise tolerated in the spacer region of DNA phosphorothioate, for example, according to EP 2742135.

Stereorandom phosphorothioate linkages

Phosphorothioate linkages are internucleoside phosphate linkages in which one of the non-bridging oxygens has been replaced by a sulphur. Substitution of sulfur for one of the non-bridging oxygens introduces a chiral center and thus, within a single phosphorothioate oligonucleotide, each phosphorothioate internucleoside linkage will be in the s (sp) or r (rp) stereoisomeric form. Such internucleoside linkages are referred to as "chiral internucleoside linkages". In contrast, phosphodiester internucleoside linkages are achiral in that they have two non-terminal oxygen atoms.

The nomenclature of stereocenter chirality is determined according to the standard Cahn-Ingold-Prelog rule (CIP precedence rule) first published in the following documents: cahn, r.s.; ingold, c.k.; prelog, V. (1966) "Specification of Molecular Chirality" Angewandte Chemie International Edition 5(4): 385-415. doi: 10.1002/anie.196603851.

The stereoselectivity of coupling and subsequent sulfurization was not controlled during standard oligonucleotide synthesis. For this reason, the stereochemistry of each phosphorothioate internucleoside linkage is randomly Sp or Rp, and therefore phosphorothioate oligonucleotides produced by conventional oligonucleotide synthesis can be as many as 2 in practice^XA variety of different phosphorothioate diastereomers exist, where X is the number of phosphorothioate internucleoside linkages. Such oligonucleotides are referred to herein as stereorandom phosphorothioate oligonucleotides and do not contain any stereodefined internucleoside linkages. Thus, a stereorandom phosphorothioate oligonucleotide is a mixture of individual diastereomers resulting from a non-stereochemically defined synthesis. In this context, a mixture is defined as up to 2^XDifferent phosphorothioate diastereomers.

Stereospecific internucleoside linkages

A stereospecific internucleoside linkage is a chiral internucleoside linkage having a diastereomeric excess in one of its two diastereomeric forms Rp or Sp.

It will be appreciated that stereoselective oligonucleotide synthesis methods used in the art typically provide a diastereoselectivity of at least about 90% or at least about 95% at each chiral internucleoside linkage, and thus up to about 10% such as about 5% of the oligonucleotide molecules may have alternative forms of diastereomer.

In some embodiments, the diastereomer ratio of each stereotactic chiral internucleoside linkage is at least about 90: 10. In some embodiments, the diastereomer ratio of each chiral internucleoside linkage is at least about 95: 5.

A stereospecific phosphorothioate linkage is a specific example of a stereospecific internucleoside linkage.

Stereospecific phosphorothioate linkages

A stereospecific phosphorothioate bond is one that has a diastereomeric excess in one of its two diastereomeric forms Rp or Sp.

The Rp and Sp configurations of the phosphorothioate internucleoside linkage are shown below

Wherein the 3'R group represents the 3' position of an adjacent nucleoside (5 'nucleoside) and the 5' R group represents the 5 'position of an adjacent nucleoside (3' nucleoside).

Herein, Rp internucleoside linkages can also be denoted as srP and Sp internucleoside linkages can be denoted as ssP.

In a particular embodiment, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 90:10 or at least 95: 5.

In some embodiments, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 97: 3. In some embodiments, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 98: 2. In some embodiments, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 99: 1.

In some embodiments, the stereotactic internucleoside linkage has the same diastereomeric form (Rp or Sp) in at least 97% (such as at least 98%, such as at least 99%) or (substantially) all of the oligonucleotide molecules present within a population of oligonucleotide molecules.

Diastereomeric purity can be measured in a model system with only achiral backbone (i.e. phosphodiester). Diastereomeric purity of each monomer can be measured, for example, by coupling the monomer with a stereotactic internucleoside linkage to the following model system "5't-po-t-po 3'". The result of this measurement will then give: HPLC can be used to isolate 5'DMTr-t-srp-t-po-t-po-t-po 3' or 5'DMTr-t-ssp-t-po-t-po 3'. Diastereomeric purity is determined by integrating the UV signals from two possible diastereomers and obtaining the ratio of these diastereomers (e.g., 98:2, 99:1, or >99: 1).

It will be appreciated that the diastereomeric purity of a particular single diastereomer (a single stereotactic oligonucleotide molecule) will vary with the coupling selectivity of the stereocenter defined at each internucleoside position and the number of stereotactic internucleoside linkages to be introduced. By way of example, if the coupling selectivity at each position is 97%, the resulting purity of a stereotactic oligonucleotide having 15 stereotactic internucleoside linkages would be 0.97¹⁵I.e. 63% of the desired diastereomer as compared to 37% of the other diastereomer. The purity of a defined diastereomer can be improved by purification (e.g. by HPLC, such as ion exchange chromatography or reverse phase chromatography) after synthesis.

In some embodiments, a stereotactic oligonucleotide refers to a population of oligonucleotides wherein at least about 40%, such as at least about 50%, of the population of oligonucleotides belongs to a desired diastereomer.

In other words, in some embodiments, a stereotactic oligonucleotide refers to a population of oligonucleotides wherein at least about 40%, such as at least about 50%, of the population of oligonucleotides consists of a desired (specified) stereotactic internucleoside linkage motif (also referred to as a stereotactic motif).

For stereotactic oligonucleotides comprising stereotactic internucleoside stereocenters and stereotactic internucleoside stereocenters, the purity of the stereotactic oligonucleotide is determined with reference to the% of the population of oligonucleotides that retains the desired stereotactic internucleoside linkage motif, calculated without taking into account stereotactic atactic linkages.

Nucleobases

The term nucleobase includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term "nucleobase" also covers modified nucleobases, which may differ from naturally occurring nucleobases, but which play a role during nucleic acid hybridization. In this context, "nucleobase" refers to naturally occurring nucleobases, such as adenine, guanine, cytosine, thymine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research, volume 45, page 2055 and in Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry suppl.371.4.1.

In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-mercapto-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazole-uracil, 2-thio-uracil, 2' -thio-thymine, inosine, diaminopurine, 6-aminopurine, 2, 6-diaminopurine, and 2-chloro-6-aminopurine.

Nucleobase moieties may be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C or U, wherein each letter may optionally include modified nucleobases with equivalent functionality. For example, in the exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.

Modified oligonucleotides

The term "modified oligonucleotide" describes an oligonucleotide comprising one or more sugar modified nucleosides and/or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term that has been used in the literature to describe oligonucleotides having modified nucleosides.

Stereospecific oligonucleotides

A stereotactic oligonucleotide is an oligonucleotide in which at least one internucleoside linkage is a stereotactic internucleoside linkage.

A stereotactic phosphorothioate oligonucleotide is an oligonucleotide in which at least one internucleoside linkage is a stereotactic phosphorothioate internucleoside linkage.

Complementarity

The term "complementarity" describes the ability of a nucleoside/nucleotide to undergo Watson-Crick base pairing. Watson-Crick base pairs are guanine (G) -cytosine (C) and adenine (A) -thymine (T)/uracil (U). It is to be understood that oligonucleotides may comprise nucleosides with modified nucleobases, e.g., 5-methylcytosine is often used instead of cytosine, and thus the term complementarity encompasses Watson Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research, volume 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry supply.371.4.1).

As used herein, the term "% complementary" refers to the proportion of nucleotides within a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide), wherein at a given position, the nucleotides are complementary to (i.e., form Watson Crick base pairs with) a contiguous nucleotide sequence of an individual nucleic acid molecule (e.g., a target nucleic acid) at the given position. The percentage is calculated by: (with the target sequence 5'-3' and oligonucleotide sequences from 3'-5' alignment) count two sequences between the formation of pairing of aligned base number, divided by the oligonucleotide in the total number of nucleotides and multiplied by 100. In this comparison, the misalignment (forming base pairs) of nucleobases/nucleotides is called mismatch. Preferably, insertions and deletions are not allowed when calculating the% complementarity of a contiguous nucleotide sequence.

The term "fully complementary" refers to 100% complementarity.

Identity of each other

As used herein, the term "identity" refers to the number of nucleotides in percentage of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide), wherein at a given position, the nucleotides are identical to the contiguous nucleotide sequence of an individual nucleic acid molecule (e.g., a target nucleic acid) at the given position (i.e., in terms of its ability to form Watson Crick base pairs with complementary nucleosides). The percentage is calculated by: the number of aligned bases that are identical between the two sequences is counted, divided by the total number of nucleotides in the oligonucleotide and multiplied by 100. Percent identity ═ match × 100)/length of the aligned region. Preferably, insertions and deletions are not allowed when calculating the% complementarity of a contiguous nucleotide sequence.

Hybridization of

As used herein, the term "hybridizing" should be understood to mean that two nucleic acid strands form hydrogen bonds between base pairs on opposing strands to form duplexes (e.g., oligonucleotides and target nucleic acids). The affinity of the binding between two nucleic acid strands is the strength of hybridization. Usually by melting temperature (T)_m) Which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. Under physiological conditions, T_mNot exactly in strict proportion to affinity (Mergny and Lacreox, 2003, Oligonucleotides 13: 515-. The standard state Gibbs free energy Δ G ° is a more accurate representation of binding affinity and dissociation constant (K) from the reaction_d) Multiplying by Δ G ° — RTln (K)_d) Where R is the gas constant and T is the absolute temperature. Thus, the very low Δ G ° of the reaction between the oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and the target nucleic acid. Ag ° is the energy associated with a reaction in which the water concentration is 1M, pH at 7 and the temperature is 37 ℃. Hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction, and Δ G ° is less than zero for the spontaneous reaction. Δ G ° can be measured experimentally, for example, by using the Isothermal Titration Calorimetry (ITC) method as described in Hansen et al, 1965, chem. Comm.36-38 and Holdgate et al, 2005, Drug Discov Today. The techniques of the artOne will know that commercial equipment can be used to measure Δ G °. Δ G can also be estimated numerically by using the nearest neighbor model as described by Santa Lucia,1998, Proc Natl Acad Sci USA.95: 1460-. In order to have the possibility of modulating its intended nucleic acid target by hybridization, for oligonucleotides of 10-30 nucleotides in length, the oligonucleotides of the invention hybridize with the target nucleic acid with an estimate of Δ G ° of less than-10 kcal. In some embodiments, the degree or intensity of hybridization is measured in terms of the standard state Gibbs free energy Δ G °. For oligonucleotides 8-30 nucleotides in length, the oligonucleotide can hybridize to the target nucleic acid with an estimate of Δ G ° of less than-10 kcal, such as less than-15 kcal, such as less than-20 kcal, and such as less than-25 kcal. In some embodiments, the oligonucleotide hybridizes to a target nucleic acid at an estimated Δ G ° value of-10 to-60 kcal, such as-12 to-40 kcal, such as from-15 to-30 kcal or-16 to-27 kcal, such as-18 to-25 kcal.

Sugar modification

Oligomers of the invention may comprise one or more nucleosides having a modified sugar moiety (i.e., a modification of the sugar moiety) when compared to the ribose sugar moiety found in DNA and RNA.

Many modified nucleosides have been prepared with ribose moieties, the primary purpose being to improve certain properties of the oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those in which the ribose ring structure is modified, for example, by substitution with: a hexose ring (HNA) or a bicyclic ring (LNA) typically having a double-base bridge between the C2 and C4 carbons on the ribose ring or a non-linked ribose ring typically lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO 2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example in the case of Peptide Nucleic Acid (PNA) or morpholino nucleic acid.

Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2' -OH group naturally present in DNA and RNA nucleosides. For example, substituents may be introduced at the 2', 3', 4 'or 5' positions.

2' sugar modified nucleosides

A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or-OH at the 2' position (a 2' substituted nucleoside) or comprising a 2' linking diradical capable of forming a bridge between the 2' carbon and the second carbon atom in the ribose ring, such as a LNA (2' -4' diradical bridged) nucleoside.

In fact, much effort has been expended to develop 2 'substituted nucleosides, and many 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2' modified sugars can provide enhanced binding affinity and/or increased nuclease resistance to oligonucleotides. Examples of 2 'substituted modified nucleosides are 2' -O-alkyl-RNA, 2 '-O-methyl-RNA, 2' -alkoxy-RNA, 2 '-O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2 '-fluoro-RNA and 2' -F-ANA nucleosides. Other examples can be found in, for example, Freier & Altmann; nucleic acid Res.,1997,25,4429-4443, Uhlmann; opinion in Drug Development,2000,3(2),293-213 and Deleavey and Damha, Chemistry and Biology 2012,19, 937. The following are schematic representations of some 2' substituted modified nucleosides.

For the present invention, the 2 'substitution does not include 2' bridged molecules such as LNA.

Locked nucleic acid nucleosides (LNA nucleosides)

An "LNA nucleoside" is a 2' -modified nucleoside comprising a diradical of C2' and C4' linking the ribose ring of the nucleoside (also referred to as a "2 ' -4' bridge"), which constrains or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or Bicyclic Nucleic Acids (BNA). When LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule, the locking of the ribose conformation is associated with an enhanced affinity for hybridization (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide/complementary duplex.

Non-limiting exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al, Bioorganic & Med.Chem.Lett.12,73-76, Seth et al J.Org.Chem.2010, Vol.75 (5), pp.1569-81 and Mitsuoka et al, Nucleic Acids Research 2009,37(4), 1225-1238.

The 2'-4' bridge contains 2 to 4 bridging atoms and has in particular the formula-X-Y-, wherein X is bound to C4 'and Y is bound to C2',

wherein

X is oxygen, sulfur or-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(＝CR^aR^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-；-O-NR^a-、-NR^a-O-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

Y is oxygen, sulfur, - (CR)^aR^b)_n-、-CR^aR^b-O-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

With the proviso that-X-Y-is not-O-O-, Si (R)^a)₂-Si(R^a)₂-、-SO₂-SO₂-、-C(R^a)＝C(R^b)-C(R^a)＝C(R^b)、-C(R^a)＝N-C(R^a)＝N-、-C(R^a)＝N-C(R^a)＝C(R^b)、-C(R^a)＝C(R^b)-C(R^a) N-or-Se-;

j is oxygen, sulfur, ═ CH₂Or ═ N (R)^a)；

R^aAnd R^bIndependently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, mercapto, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, ureido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, mercaptoalkylthio, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC (═ X^a)R^c、-OC(＝X^a)NR^cR^dand-NR^eC(＝X^a)NR^cR^d；

Or two geminal R^aAnd R^bTogether form an optionally substituted methylene group;

or two geminal R^aAnd R^bTogether with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl group, and only one carbon atom in-X-Y-has this condition;

wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy, and substituted methylene are alkyl, alkenyl, alkynyl, and methylene substituted with 1 to 3 substituents independently selected from the group consisting of: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl and heteroaryl;

X^ais oxygen, sulfur or-NR^c；

R^c、R^dAnd R^eIndependently selected from hydrogen and alkyl; and is

n is 1,2 or 3.

In another embodiment of the invention, X is oxygen, sulfur, -NR^a-、-CR^aR^b-or-C (═ CR)^aR^b) -, especially oxygen, sulfur, -NH-, -CH₂-or-C (═ CH)₂) -, more particularly oxygen.

In another embodiment of the present invention, Y is-CR^aR^b-、-CR^aR^b-CR^aR^b-or-CR^aR^b-CR^aR^b-CR^aR^b-, in particular-CH₂-CHCH₃-、-CHCH₃-CH₂-、-CH₂-CH₂-or-CH₂-CH₂-CH₂-。

In a particular embodiment of the invention, -X-Y-is-O- (CR)^aR^b)_n-、-S-CR^aR^b-、-N(R^a)CR^aR^b-、-CR^aR^b-CR^aR^b-、-O-CR^aR^b-O-CR^aR^b-、-CR^aR^b-O-CR^aR^b-、-C(＝CR^aR^b)-CR^aR^b-、-N(R^a)CR^aR^b-、-O-N(R^a)-CR^aR^b-or-N (R)^a)-O-CR^aR^b-。

In a particular embodiment of the invention, R^aAnd R^bIndependently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl and alkoxyalkyl, in particular hydrogen, halogen, alkyl and alkoxyalkyl.

In another embodiment of the invention, R^aAnd R^bIndependently selected from hydrogen, fluoro, hydroxy, methyl and-CH₂-O-CH₃In particular hydrogen, fluoro, methyl and-CH₂-O-CH₃Group (d) of (a).

Advantageously R of-X-Y-^aAnd R^bOne of which is as defined above and the others are both hydrogen.

In another embodiment of the invention, R^aHydrogen or alkyl, in particular hydrogen or methyl.

In another embodiment of the invention, R^bHydrogen or alkyl, in particular hydrogen or methyl.

In a particular embodiment of the invention, R^aAnd R^bOne or both of which are hydrogen.

In a particular embodiment of the invention, R^aAnd R^bOnly one of which is hydrogen.

In a particular embodiment of the invention, R^aAnd R^bOne of them is methyl and the other is hydrogen.

In a particular embodiment of the invention, R^aAnd R^bAnd are both methyl.

In a particular embodiment of the invention, -X-Y-is-O-CH₂-、-S-CH₂-、-S-CH(CH₃)-、-NH-CH₂-、-O-CH₂CH₂-、-O-CH(CH₂-O-CH₃)-、-O-CH(CH₂CH₃)-、-O-CH(CH₃)-、-O-CH_2-O-CH₂-、-O-CH₂-O-CH₂-、-CH₂-O-CH₂-、-C(＝CH₂)CH₂-、-C(＝CH₂)CH(CH₃)-、-N(OCH₃)CH₂-or-N (CH)₃)CH₂-；

In a particular embodiment of the invention, -X-Y-is-O-CR^aR^b-, wherein R^aAnd R^bIndependently selected from hydrogen, alkyl and alkoxyalkyl, especially hydrogen, methyl and-CH₂-O-CH₃Group (d) of (a).

In one embodiment, -X-Y-is-O-CH₂-or-O-CH (CH)₃) -, especially-O-CH₂-。

The 2'-4' bridge may be located below the plane of the ribose ring (beta-D-configuration), or above the plane of the ring (alpha-L-configuration), as shown in formulas (A) and (B), respectively.

The LNA nucleosides according to the invention are in particular of the formula (B1) or (B2)

Wherein

W is oxygen, sulfur, -N (R)^a) -or-CR^aR^b-, especially oxygen;

b is a nucleobase or a modified nucleobase;

z is an internucleoside linkage to an adjacent nucleoside or a 5' terminal group;

z is an internucleoside linkage to an adjacent nucleoside or a 3' terminal group;

R¹、R²、R³、R⁵and R^5*Independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkoxyalkyl, azido, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, and aryl; and is

X、Y、R^aAnd R^bAs defined above.

In one embodiment, in the definition of-X-Y-, R^aHydrogen or alkyl, in particular hydrogen or methyl. In another embodiment, in the definition of-X-Y-, R^bHydrogen or alkyl, in particular hydrogen or methyl. In another embodiment, in the definition of-X-Y-, R^aAnd R^bOne or both of which are hydrogen. In one embodiment, in the definition of-X-Y-, R^aAnd R^bOnly one of which is hydrogen. In one embodiment, in the definition of-X-Y-, R^aAnd R^bOne of which is methyl and the other is hydrogen. In one embodiment, in the definition of-X-Y-, R^aAnd R^bAnd are both methyl.

In another embodiment, in the definition of X, R^aHydrogen or alkyl, in particular hydrogen or methyl. In another embodiment, in the definition of X, R^bHydrogen or alkyl, in particular hydrogen or methyl. In a particular embodiment, in the definition of X, R^aAnd R^bOne or both of which are hydrogen. In a particular embodiment, in the definition of X, R^aAnd R^bOnly one of which is hydrogen. In a particular embodiment, in the definition of X, R^aAnd R^bOne of which is methyl and the other is hydrogen. In one embodiment, in the definition of X，R^aAnd R^bAnd are both methyl.

In another embodiment, in the definition of Y, R^aHydrogen or alkyl, in particular hydrogen or methyl. In another embodiment, in the definition of Y, R^bHydrogen or alkyl, in particular hydrogen or methyl. In one embodiment, in the definition of Y, R^aAnd R^bOne or both of which are hydrogen. In one embodiment, in the definition of Y, R^aAnd R^bOnly one of which is hydrogen. In one embodiment, in the definition of Y, R^aAnd R^bOne of which is methyl and the other is hydrogen. In one embodiment, in the definition of Y, R^aAnd R^bAnd are both methyl.

In a particular embodiment of the invention, R¹、R²、R³、R⁵And R^5*Independently selected from hydrogen and alkyl, especially hydrogen and methyl.

In another particularly advantageous embodiment of the invention, R¹、R²、R³、R⁵And R^5*And are both hydrogen.

In another embodiment of the invention, R¹、R²、R³Are simultaneously all hydrogen, R⁵And R^5*One of which is hydrogen and the other is as defined above, in particular alkyl, more in particular methyl.

In a particular embodiment of the invention, R⁵And R^5*Independently selected from hydrogen, halogen, alkyl, alkoxyalkyl and azido, in particular from hydrogen, fluoro, methyl, methoxyethyl and azido. In particular, in an advantageous embodiment of the invention, R⁵And R^5*One of which is hydrogen and the other is alkyl, in particular methyl, halogen, in particular fluoro, alkoxyalkyl, in particular methoxyethyl or azido; or R⁵And R^5*And are both hydrogen or halogen, in particular both hydrogen and fluoro. In such embodiments, W may advantageously be oxygen, and-X-Y-is advantageously-O-CH₂-。

In a particular embodiment of the invention, -X-Y-is-O-CH₂-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352 and WO 2004/046160 (all of which are incorporated herein by reference) and include β -D-oxy LNA and α -L-oxy LNA nucleosides generally known in the art.

In another embodiment of the invention, -X-Y-is-S-CH₂-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such thiolan nucleosides are disclosed in WO 99/014226 and WO 2004/046160 (which are hereby incorporated by reference).

In another embodiment of the invention, -X-Y-is-NH-CH₂-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such aminolna nucleosides are disclosed in WO 99/014226 and WO 2004/046160 (which are incorporated herein by reference).

In another embodiment of the invention, -X-Y-is-O-CH₂CH₂-or-OCH₂CH₂CH₂-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such LNA nucleosides are disclosed in WO 00/047599 and Morita et al, Bioorganic&Med, chem, lett, 12,73-76 (these documents are incorporated herein by reference) and include 2'-O-4' C-ethylene bridged nucleic acids (ENA) as generally known in the art.

In another embodiment of the invention, -X-Y-is-O-CH₂-, W is oxygen, R¹、R²、R³Are simultaneously all hydrogen, R⁵And R^5*One of which is hydrogen and the other is not hydrogen, such as alkyl, for example methyl. Such 5' substituted LNA nucleosides are disclosed in WO 2007/134181 (which is hereby incorporated by reference).

In another embodiment of the invention, -X-Y-is-O-CR^aR^b-, wherein R^aAnd R^bOne or both of which are not hydrogen, in particular alkyl, such as methyl, W is oxygen, R¹、R²、R³Are simultaneously all hydrogen, R⁵And R^5*One of which is hydrogen and the other is not hydrogen, in particular an alkyl group, for example methyl. Such doubly modified LNA nucleosides are disclosed in WO 2010/077578 (which is hereby incorporated by reference).

In another embodiment of the invention, -X-Y-is-O-CHR^a-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such 6' substituted LNA nucleosides are disclosed in WO 2010/036698 and WO 2007/090071 (both of which are incorporated herein by reference). In such 6' -substituted LNA nucleosides, R^aIn particular C₁-C₆Alkyl groups such as methyl.

In another embodiment of the invention, -X-Y-is-O-CH (CH)₂-O-CH₃) - ("2' O-methoxyethyl bicyclic nucleic acid") (Seth et al, J.org.chem.2010, Vol.75 (5), p.1569-81).

In another embodiment of the invention, -X-Y-is-O-CH (CH)₂CH₃)-。

In another embodiment of the invention, -X-Y-is-O-CH (CH)₂-O-CH₃) -, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such LNA nucleosides are also known in the art as cyclic moes (cmoe) and are disclosed in WO 2007/090071.

In another embodiment of the invention, -X-Y-is-O-CH (CH)₃) - ("2' O-ethylbicyclic nucleic acid") (Seth et al, J.org.chem.2010, Vol.75 (5), pp.1569-81).

In another embodiment of the invention, -X-Y-is-O-CH_2-O-CH₂- (Seth et al, j. org. chem2010, supra).

In another embodiment of the invention, -X-Y-is-O-CH (CH)₃) -, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such 6' -methyl LNA nucleosides are also known in the art as cET nucleosides and can be (S) -cET or (R) -cET diastereomers, as disclosed in WO 2007/090071(β -D) and WO 2010/036698(α -L), both of which are incorporated herein by reference.

In another embodiment of the invention, -X-Y-is-O-CR^aR^b-, wherein R^aAnd R^bAre not all hydrogen, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. In one embodiment, R^aAnd R^bBoth are alkyl radicals, in particular both are methyl radicals. Such 6' -di-substituted LNA nucleosides are disclosed in WO 2009/006478 (which is hereby incorporated by reference).

In another embodiment of the invention, -X-Y-is-S-CHR^a-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. Such 6' -substituted thiolna nucleosides are disclosed in WO2011/156202 (which is hereby incorporated by reference). In specific embodiments of such 6' -substituted thioLNAs, R^aIs an alkyl group, in particular methyl.

In a particular embodiment of the invention, -X-Y-is-C (═ CH)₂)C(R^aR^b)-、-C(＝CHF)C(R^aR^b) -or-C (═ CF)₂)C(R^aR^b) -, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. R^aAnd R^bAdvantageously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluoro and methoxymethyl. R^aAnd R^bIn particular both hydrogen and methyl, or R^aAnd R^bOne of which is hydrogen and the other is methyl. Such vinyl carbon LNA nucleosides are disclosed in WO 2008/154401 and WO 2009/067647 (both of which are incorporated herein by reference).

In a particular embodiment of the invention, -X-Y-is-N (OR)^a)-CH₂-, W is oxygen,and R is¹、R²、R³、R⁵And R^5*And are both hydrogen. In one embodiment, R^aIs an alkyl group such as methyl. Such LNA nucleosides are also known as N-substituted LNAs and are disclosed in WO 2008/150729 (which is hereby incorporated by reference).

In a particular embodiment of the invention, -X-Y-is-O-N (R)^a)-、-N(R^a)-O-、-NR^a-CR^aR^b-CR^aR^b-or-NR^a-CR^aR^b-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. R^aAnd R^bAdvantageously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluoro and methoxymethyl. In one embodiment, R^aIs alkyl, such as methyl, R^bHydrogen or methyl, in particular hydrogen (Seth et al, j. org. chem2010, supra).

In a particular embodiment of the invention, -X-Y-is-O-N (CH)₃) - (Seth et al, j.org.chem2010, supra).

In a particular embodiment of the invention, R⁵And R^5*And are both hydrogen. In another embodiment of the invention, R⁵And R^5*One of which is hydrogen and the other is an alkyl group, such as methyl. In such embodiments, R¹、R²And R³May in particular be hydrogen, and-X-Y-may in particular be-O-CH₂-or-O-CHC (R)^a)₃-, such as-O-CH (CH)₃)-。

In a particular embodiment of the invention, -X-Y-is-CR^aR^b-O-CR^aR^b-, such as-CH₂-O-CH₂-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. In such embodiments, R^aMay in particular be alkyl, such as methyl, R^bHydrogen or methyl, especially hydrogen. Such LNA nucleosides are also known as conformationally constrained nucleosides (CRNs) and are disclosed in WO 2013/036868 (which is herein incorporated by reference)Incorporated by reference).

In a particular embodiment of the invention, -X-Y-is-O-CR^aR^b-O-CR^aR^b-, such as-O-CH₂-O-CH₂-, W is oxygen, and R¹、R²、R³、R⁵And R^5*And are both hydrogen. R^aAnd R^bAdvantageously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluoro and methoxymethyl. In such embodiments, R^aMay be an alkyl group, such as methyl, R^bHydrogen or methyl, especially hydrogen. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al, Nucleic Acids Research 2009,37(4),1225-1238, which is hereby incorporated by reference.

It will be appreciated that LNA nucleosides can be either β -D or α -L stereoisomers unless specified.

Specific examples of LNA nucleosides of the invention are presented in scheme 1 (where B is as defined above).

Scheme 1

Specific LNA nucleosides are β -D-oxy-LNA, 6 '-methyl- β -D-oxy-LNA such as (S) -6' -methyl- β -D-oxy-LNA ((S) -cET) and ENA.

RNase H activity and recruitment

The RNase H activity of the antisense oligonucleotide refers to its ability to recruit RNase H when it forms duplexes with complementary RNA molecules. WO01/23613 provides in vitro methods for determining RNaseH activity, which can be used to determine the ability to recruit RNaseH. An oligonucleotide is generally considered to be capable of recruiting RNase H if it has an initial rate of at least 5% (such as at least 10% or more than 20%) of the initial rate (in pmol/l/min) when provided with a complementary target nucleic acid sequence determined using: oligonucleotides having the same base sequence as the modified oligonucleotides tested but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide were used, and the methods provided in examples 91-95 of WO01/23613 (incorporated herein by reference) were used. For use in determining the RRNase H activity, recombinant human RNase H1 was obtained from Lubio Science GmbH, Lucerne, Switzerland.

Gapmer

The antisense oligonucleotide of the invention or a contiguous nucleotide sequence thereof may be a gapmer. Antisense gapmers are commonly used to inhibit target nucleic acids by RNase H mediated degradation. A gapmer oligonucleotide comprises at least three different structural regions, namely a 5' -flank in the ' 5- >3' direction, a gap and a 3' flank F-G-F '. The "gap" region (G) comprises a contiguous DNA nucleotide which enables the oligonucleotide to recruit RNase H. The notch region is flanked by a 5' flanking region (F) comprising one or more sugar-modified nucleosides (preferably high affinity sugar-modified nucleosides) and a 3' flanking region (F ') comprising one or more sugar-modified nucleosides (preferably high affinity sugar-modified nucleosides). One or more sugar modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., the affinity enhanced sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in regions F and F 'are 2' sugar modified nucleosides, such as high affinity 2 'sugar modifications, such as independently selected from LNA and 2' -MOE.

In the gapmer design, the 5' and 3' endmost nucleosides of the gapped region are DNA nucleosides, located near the sugar-modified nucleosides of the 5' (F) or 3' (F ') regions, respectively. These flanking regions may be further defined by nucleosides having at least one sugar modification at the end furthest from the notch region (i.e., at the 5 'end of the 5' flanking region and at the 3 'end of the 3' flanking region).

The region F-G-F' forms a contiguous nucleotide sequence. The antisense oligonucleotides of the invention or contiguous nucleotide sequences thereof may comprise a gapmer region of the formula F-G-F'.

The total length of the gapmer design F-G-F' may be, for example, 12 to 32 nucleosides, such as 13 to 24 nucleosides, such as 14 to 22 nucleosides, such as 14 to 17 nucleosides, such as 16 to 18 nucleosides.

For example, the gapmer oligonucleotides of the invention can be represented by the formula:

F_1-8-G_5-16-F'_1-8such as

F_1-8-G_7-16-F'_2-8

Provided that the total length of the gapmer region F-G-F' is at least 12, such as at least 14 nucleotides.

Regions F, G and F 'are further defined below and can be incorporated into the F-G-F' formula.

Gapmer-region G

Region G of the gapmer (the gapped region) is a region of nucleosides (usually DNA nucleosides) that enables the recruitment of an oligonucleotide to an RNaseH such as human RNase H1. RNaseH is a cellular enzyme that recognizes duplexes between DNA and RNA and enzymatically cleaves RNA molecules. Suitable gapped mers may have a gapped region (G) of at least 5 or 6 consecutive DNA nucleosides in length, such as 5-16 consecutive DNA nucleosides, such as 6-15 consecutive DNA nucleosides, such as 7-14 consecutive DNA nucleosides, such as 8-12 consecutive DNA nucleotides, such as 8-12 consecutive DNA nucleosides. In some embodiments, the gap region G can consist of 6, 7, 8, 9,10,11, 12, 13, 14, 15, or 16 consecutive DNA nucleosides. In some cases, cytosine (C) DNA in the notch region may be methylated, such residues being labeled 5-methyl-cytosine (ii)^meC or replacing C with e). If cg dinucleotides are present in the gap, methylation of cytosine DNA in the gap is beneficial in reducing potential toxicity, and this modification has no significant effect on the efficacy of the oligonucleotide.

In some embodiments, the gap region G can consist of 6, 7, 8, 9,10,11, 12, 13, 14, 15, or 16 consecutive phosphorothioate-linked DNA nucleosides. In some embodiments, all internucleoside linkages in the nick are phosphorothioate linkages.

Although conventional gapmer have a DNA gap region, there are many examples of modified nucleosides that can recruit RNaseH when used within the gap region. Modified nucleosides that have been reported to recruit RNaseH when included in the notch region include, for example, α -L-LNA, C4' alkylated DNA (as described in PCT/EP2009/050349 and Vester et al, bioorg.med.chem.lett.18(2008) 2296-. UNA is a non-locked nucleic acid, typically in which the bond between C2 and C3 of the ribose has been removed, forming a non-locked "sugar" residue. The nucleoside used for modification in such a gapmer may be a nucleoside that adopts a 2' endo (DNA-like) structure when introduced into the gapped region, i.e., a modification that allows RNaseH recruitment). In some embodiments, the DNA gap region (G) described herein can optionally contain 1 to 3 sugar-modified nucleosides that adopt a 2' endo (DNA-like) structure when the gap region is introduced.

Region G- "gap destroyer"

Alternatively, there are many reports of insertion modified nucleosides that confer 3' internal conformation to the notch region of the gapmer while retaining some RNaseH activity. Such gapmer having a gap region comprising one or more 3' endo-modified nucleosides is referred to as a "gap breaker" or "gap-disrupted" gapmer, see, e.g., WO 2013/022984. The notch breaker oligonucleotide retains sufficient DNA nucleotide region within the notch region to allow recruitment of RNaseH. The ability of notch breaker oligonucleotides to design recruits RNaseH is generally sequence-specific or even compound-specific, see Rukov et al 2015Nucl. acids Res. Vol.43, page 8476-8487, which discloses "notch breaker" oligonucleotides that recruit RNaseH that in some cases provide more specific cleavage of target RNA. The modified nucleoside used in the notch region of the notch interrupter oligonucleotide may for example be a modified nucleoside conferring a 3' endo configuration, such as a 2' -O-methyl (OMe) or 2' -O-moe (moe) nucleoside, or a β -DLNA nucleoside (the bridge between C2' and C4' of the ribose ring of the nucleoside is in the β conformation), such as a β -D-oxy LNA or ScET nucleoside.

Like the gapmer comprising region G above, the gapped region of the gapmer or gapmer interfering with the gapping has a DNA nucleoside at the 5' end of the gap (adjacent to the 3' nucleoside of region F) and a DNA nucleoside at the 3' end of the gap (adjacent to the 5' nucleoside of region F '). A gapmer comprising a disruption will generally retain a region of at least 3 or 4 contiguous DNA nucleosides at the 5 'end or 3' end of the gapped region.

Exemplary designs of gap interruptor oligonucleotides include

F_1-8-[D_3-4-E₁-D_3-4]_-F'_1-8

F_1-8-[D_1-4-E₁-D_3-4]-F'_1-8

F_1-8-[D_3-4-E₁-D_1-4]-F'_1-8

Wherein the region G is in the bracket [ D ]_n-E_r-D_m]Within, D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (gap interruptor or gap interrupting nucleoside), and F 'are flanking regions as defined herein, and with the proviso that the overall length of the gapmer region F-G-F' is at least 12, such as at least 14 nucleotides in length.

In some embodiments, region G of the nick-disrupting gapmer comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9,10,11, 12, 13, 14, 15, or 16 DNA nucleosides. As described above, the DNA nucleoside may be continuous or optionally interspersed with one or more modified nucleosides, provided that the gap region G is capable of mediating RNaseH recruitment.

Gapmer-flanking region, F and F'

Region F is immediately adjacent to the 5' DNA nucleotides of region G. The 3 'endmost nucleoside of region F is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2' substituted nucleoside, such as a MOE nucleoside or a LNA nucleoside.

Region F 'is immediately adjacent to the 3' DNA nucleotides of region G. The 5' endmost nucleoside of region F ' is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2' substituted nucleoside, such as a MOE nucleoside or a LNA nucleoside.

Region F is 1-8 contiguous nucleotides in length, such as 2-6, such as 3-4 contiguous nucleotides in length. Advantageously, the 5' endmost nucleoside of region F is a sugar modified nucleoside. In some embodiments, the two 5' endmost nucleosides of region F are sugar modified nucleosides. In some embodiments, the 5' endmost nucleoside of region F is an LNA nucleoside. In some embodiments, the two 5' endmost nucleosides of region F are LNA nucleosides. In some embodiments, the two 5' endmost nucleosides of region F are 2' substituted nucleosides, such as two 3' MOE nucleosides. In some embodiments, the 5 'endmost nucleoside of region F is a 2' substituted nucleoside, such as a MOE nucleoside.

The region F' is 2-8 contiguous nucleotides in length, such as 3-6, such as 4-5 contiguous nucleotides in length. Preferably, in embodiments, the 3 'endmost nucleoside of region F' is a sugar modified nucleoside. In some embodiments, the two 3' endmost nucleosides of region F are sugar modified nucleosides. In some embodiments, the two 3' endmost nucleosides of region F are LNA nucleosides. In some embodiments, the 3' endmost nucleoside of region F is an LNA nucleoside. In some embodiments, the two 3' endmost nucleosides of region F are 2' substituted nucleosides, such as two 3' MOE nucleosides. In some embodiments, the 3 'endmost nucleoside of region F is a 2' substituted nucleoside, such as a MOE nucleoside.

It should be noted that when the length of region F or F' is one, it is preferably an LNA nucleoside.

In some embodiments, regions F and F' independently consist of or comprise a contiguous sequence of sugar modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F can be independently selected from the group consisting of 2 '-O-alkyl-RNA units, 2' -O-methyl-RNA, 2 '-amino-DNA units, 2' -fluoro-DNA units, 2 '-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units, and 2' -fluoro-ANA units.

In some embodiments, regions F and F 'independently comprise both LNA and 2' substituted modified nucleosides (hybrid wing design).

In some embodiments, regions F and F' consist of only one type of sugar modified nucleoside, such as only MOE or only β -D-oxylna or only ScET. Such designs are also referred to as homogeneous flap or homogeneous gapmer designs.

In some embodiments, all nucleosides of region F or F 'or F and F' are LNA nucleosides, such as independently selected from β -D-oxy LNA, ENA or ScET nucleosides. In some embodiments, region F consists of 1-5, such as 2-4, such as 3-4, such as 1,2, 3, 4, or 5 consecutive LNA nucleosides. In some embodiments, all nucleosides of regions F and F' are β -D-oxy LNA nucleosides.

In some embodiments, all nucleosides of region F or F ' or F and F ' are 2' substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments, region F consists of 1,2, 3, 4,5, 6, 7, or 8 consecutive OMe or MOE nucleosides. In some embodiments, only one flanking region may consist of a 2' substituted nucleoside, such as an OMe or MOE nucleoside. In some embodiments, the 5'(F') flanking region consists of a 2 'substituted nucleoside, such as an OMe or MOE nucleoside, while the 3' (F) flanking region comprises at least one LNA nucleoside, such as a β -D-oxy LNA nucleoside or an cET nucleoside. In some embodiments, the 3'(F') flanking region consists of a 2 'substituted nucleoside, such as an OMe or MOE nucleoside, while the 5' (F) flanking region comprises at least one LNA nucleoside, such as a β -D-oxy LNA nucleoside or an cET nucleoside.

In some embodiments, all modified nucleosides of regions F and F ' are LNA nucleosides, such as independently selected from β -D-oxy LNA, ENA or ScET nucleosides, wherein region F or F ' or F and F ' may optionally comprise DNA nucleosides (alternating flanking, see for more detail, these definitions). In some embodiments, all modified nucleosides of regions F and F ' are β -D-oxolna nucleosides, wherein region F or F ' or F and F ' may optionally comprise DNA nucleosides (alternating flanking, see these for more detail).

In some embodiments, the 5' and 3' endmost nucleosides of regions F and F ' are LNA nucleosides, such as β -D-oxy LNA nucleosides or ScET nucleosides.

In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F' and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between nucleosides of regions F or F ', F and F' is a phosphorothioate internucleoside linkage.

Other notch polymer designs are disclosed in WO 2004/046160, WO 2007/146511 and WO 2008/113832, which are hereby incorporated by reference.

LNA gapmers

An LNA gapmer is one in which one or both of regions F and F' comprise or consist of LNA nucleosides. A β -D-oxygapmer is a gapmer in which one or both of regions F and F' comprise or consist of β -D-oxylna nucleosides.

In some embodiments, the LNA gapmer has the formula: [ LNA]_1-5- [ region G]-[LNA]_1-5Wherein region G is as defined in the definition of gapmer region G.

MOE gapped mers

A MOE gapmer is one in which regions F and F' are composed of MOE nucleosides. In some embodiments, the MOE gapmer is designed as [ MOE]_1-8- [ region G]-[MOE]_1-8Such as [ MOE]_2-7- [ region G]_5-16-[MOE]_2-7Such as [ MOE]_3-6- [ region G]-[MOE]_3-6Wherein region G is as defined in the definition of gapmer. MOE gapmers having the 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

Hybrid wingtip notch polymer

The mixed-mode flanking gapmers are LNA gapmers wherein one or both of region F and region F ' comprise 2' substituted nucleosides, such as 2' substituted nucleosides independently selected from the group consisting of: 2 '-O-alkyl-RNA units, 2' -O-methyl-RNA, 2 '-amino-DNA units, 2' -fluoro-DNA units, 2 '-alkoxy-RNA, MOE units, arabinonucleic acid (ANA) units and 2' -fluoro-ANA units such as MOE nucleosides. In some embodiments, wherein at least one of regions F and F ' or both regions F and F ' comprise at least one LNA nucleoside, the remaining nucleosides of regions F and F ' are independently selected from the group consisting of MOE and LNA. In some embodiments, wherein at least one of region F or F ' or both regions F and F ' comprise at least two LNA nucleosides, the remaining nucleosides of regions F and F ' are independently selected from the group consisting of MOE and LNA. In some hybrid wing embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides.

Hybrid flanking notch-mer designs are disclosed in WO 2008/049085 and WO 2012/109395 (both of which are incorporated herein by reference).

Alternating flanking gapmer

The flanking region may comprise both LNA and DNA nucleosides and is referred to as an "alternating flank" because it comprises alternating motifs of LNA-DNA-LNA nucleosides. Notch mers comprising such alternating flanks are referred to as "alternating flank notch mers". Thus, an "alternating flanking gapmer" is an LNA gapmer oligonucleotide, wherein at least one flank (F or F') comprises DNA in addition to LNA nucleosides. In some embodiments, at least one or both of regions F or F' comprises both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking regions F or F ', or both F and F ', comprise at least three nucleosides, wherein the 5' and 3' endmost nucleosides of the F and/or F ' region are LNA nucleosides.

Alternative flanking LNA gapmers are disclosed in WO 2016/127002.

The alternating flanking regions may comprise up to 3 consecutive DNA nucleosides, such as 1 to 2 or 1 or 2 or 3 consecutive DNA nucleosides.

The alternating flanks may be annotated as a series of integers representing a number of LNA nucleosides (L) followed by a number of DNA nucleosides (D), e.g.

[L]_1-3-[D]_1-4-[L]_1-3

[L]_1-2-[D]_1-2-[L]_1-2-[D]_1-2-[L]_1-2

In oligonucleotide design, these are usually expressed as numbers, so that 2-2-1 represents 5' [ L ]]₂-[D]₂-[L]3', and 1-1-1-1-1 represents 5' [ L ]]-[D]-[L]-[D]-[L]3'. The length of the flanks (regions F and F') in the oligonucleotide with alternating flanks may independently be 3 to 10 nucleosides, such as 4 to 8, such as 5 to 6 nucleosides, such as 4,5, 6 or 7 modified nucleosides. In some embodiments, only one flank of the gapmer oligonucleotide is alternating, while the other flanks consist of LNA nucleotides. It may be advantageous to have at least two LNA nucleosides at the 3' end of the 3' flank (F ') to confer additional exonuclease resistance. Some examples of oligonucleotides with alternating flanks are:

[L]_1-5-[D]_1-4-[L]_1-3-[G]_5-16-[L]_2-6

[L]_1-2-[D]_1-2-[L]_1-2-[D]_1-2-[L]_1-2-[G]_5-16-[L]_1-2-[D]_1-3-[L]_2-4

[L]_1-5-[G]_5-16-[L]-[D]-[L]-[D]-[L]₂

provided that the overall length of the gapmer is at least 12, such as at least 14 nucleotides.

Region D 'or D' in the oligonucleotide "

An oligonucleotide of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide that is complementary to the target nucleic acid, such as the gapmer F-G-F ', and other 5' and/or 3' nucleosides. The additional 5 'and/or 3' nucleosides can be fully complementary to the target nucleic acid or not. Such other 5' and/or 3' nucleosides may be referred to herein as regions D ' and D ".

The addition region D' or D "may be used for the purpose of joining a contiguous nucleotide sequence (such as a gapmer) to a conjugate moiety or another functional group. When used to join a contiguous nucleotide sequence to a conjugate moiety, it can be used as a biologically cleavable linker. Alternatively, it may be used to provide exonuclease protection or to facilitate synthesis or manufacture.

The regions D ' and D "can be attached to the 5' end of region F or the 3' end of region F ', respectively, to yield the following formulas D ' -F-G-F ', F-G-F ' -D", or

Designing D ' -F-G-F ' -D '. In this case, F-G-F 'is the gapmer portion of the oligonucleotide, and region D' or D "constitutes a separate part of the oligonucleotide.

The regions D' or D "may independently comprise or consist of 1,2, 3, 4 or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar modified nucleotides such as DNA or RNA or base modified versions of these. The D' or D "region can be used as a nuclease-sensitive, biologically cleavable linker (see definition of linker). In some embodiments, the additional 5 'and/or 3' terminal nucleotide is linked to a phosphodiester linkage and is DNA or RNA. Nucleotide-based bio-cleavable linkers suitable for use as region D' or D "are disclosed in WO 2014/076195, which include by way of example phosphodiester linked DNA dinucleotides. The use of biologically cleavable linkers in multi-oligonucleotide constructs is disclosed in WO 2015/113922, where they are used to link multiple antisense constructs (e.g. gapmer regions) within a single oligonucleotide.

In one embodiment, the oligonucleotide of the invention comprises regions D' and/or D "in addition to the contiguous nucleotide sequence constituting the gapmer.

In some embodiments, the oligonucleotides of the invention can be represented by the formula:

F-G-F', in particular F_1-8-G_5-16-F'_2-8

D ' -F-G-F ', in particular D '_1-3-F_1-8-G_5-16-F'_2-8

F-G-F '-D', in particular F_1-8-G_5-16-F'_2-8-D”_1-3

D '-F-G-F' -D ', especially D'_1-3-F_1-8-G_5-16-F'_2-8-D”_1-3

In some embodiments, the internucleoside linkage between region D' and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage between region F' and region D "is a phosphodiester linkage.

Whole polymer

In some embodiments, all of the nucleotides of an oligonucleotide or a contiguous nucleotide sequence thereof are sugar modified nucleotides. Such oligonucleotides are referred to herein as whole polymers.

In some embodiments, all of the sugar modified nucleosides of the multimer comprise the same sugar modification, e.g., they may all be LNA nucleosides, or may all be 2' O-MOE nucleosides. In some embodiments, the sugar modified nucleosides of the polypolymer can be independently selected from LNA nucleosides and 2 'substituted nucleosides, such as 2' substituted nucleosides selected from the group consisting of: 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2' -fluoro-RNA and 2' -F-ANA nucleosides. In some embodiments, the oligonucleotide comprises a LNA nucleoside and a 2 'substituted nucleoside, such as a 2' substituted nucleoside selected from the group consisting of: 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2' -fluoro-RNA and 2' -F-ANA nucleosides. In some embodiments, the oligonucleotide comprises an LNA nucleoside and a 2' -O-MOE nucleoside. In some embodiments, the oligonucleotide comprises a (S) cET LNA nucleoside and a 2' -O-MOE nucleoside. In some embodiments, each nucleoside unit of the oligonucleotide is a 2' substituted nucleoside. In some embodiments, each nucleoside unit of the oligonucleotide is a 2' -O-MOE nucleoside.

In some embodiments, all of the nucleotides of an oligonucleotide or a contiguous nucleotide sequence thereof are LNA nucleotides, such as β -D-oxy-LNA nucleotides and/or (S) cET nucleotides. In some embodiments, such LNA whole-mer oligonucleotides are between 7 and 12 nucleosides in length (see, e.g., WO 2009/043353). Such short intact LNA oligonucleotides are particularly effective in inhibiting micrornas.

Many holopolymer compounds are very effective as therapeutic oligomers, particularly when targeting micrornas (antimir) or as Splice Switching Oligomers (SSO).

In some embodiments, the holopolymer comprises or consists of at least one XYX or YXY sequence motif such as the repeat sequence XYX or YXY, where X is LNA and Y is a substitute (i.e., non-LNA) nucleotide analog, such as a 2'-OMe RNA unit and a 2' -fluoro DNA unit. In some embodiments, the above sequence motif can be, for example, XXY, XYX, YXY, or YYX.

In some embodiments, a full polymer may comprise or consist of a contiguous nucleotide sequence of between 7 and 24 nucleotides, such as 7, 9,10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

In some embodiments, the contiguous nucleotide sequence of the full polymer comprises at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as 95%, such as 100% LNA units. For full LNA compounds, it is advantageous that they are less than 12, such as 7-10 nucleotides in length.

The remaining units may be selected from the non-LNA nucleotide analogues mentioned herein, such as those selected from the group consisting of: 2' -O-alkyl-RNA units, 2' -OMe-RNA units, 2' -amino-DNA units, 2' -fluoro-DNA units, LNA units, PNA units, HNA units, INA units and 2' MOE RNA units or 2' -OMe RNA units and 2' -fluoro-DNA units.

Mixed polymer

The term "heteropolymer" refers to an oligomer comprising both DNA nucleosides and sugar-modified nucleosides, wherein there are contiguous DNA nucleosides of insufficient length that recruit RNaseH. Suitable interpolymers may contain up to 3 or up to 4 consecutive DNA nucleosides. In some embodiments, the heteropolymer, or contiguous nucleotide sequence thereof, comprises alternating sugar modified nucleotide regions and DNA nucleotide regions. Oligonucleotides that do not recruit RNaseH can be prepared by incorporating alternating regions of sugar-modified nucleosides that form an RNA-like (3' endo) conformation with a short DNA nucleoside region when the oligonucleotide is incorporated. Advantageously, the sugar-modified nucleoside is an affinity-enhancing sugar-modified nucleoside.

Oligonucleotide hybrids are commonly used to provide occupancy-based regulation of target genes, such as splice regulators or microrna inhibitors.

In some embodiments, the sugar-modified nucleotides in the mixed polymer or contiguous nucleotide sequence thereof comprise or are all LNA nucleotides, such as (S) cET or β -D-oxolna nucleotides.

In some embodiments, all of the sugar modified nucleosides of the cocktail comprise the same sugar modification, e.g., they can all be LNA nucleosides, or can all be 2' O-MOE nucleosides. In some embodiments, the sugar modified nucleosides of the cocktail can be independently selected from LNA nucleosides and 2 'substituted nucleosides, such as 2' substituted nucleosides selected from the group consisting of: 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2' -fluoro-RNA and 2' -F-ANA nucleosides. In some embodiments, the oligonucleotide comprises a LNA nucleoside and a 2 'substituted nucleoside, such as a 2' substituted nucleoside selected from the group consisting of: 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2' -fluoro-RNA and 2' -F-ANA nucleosides. In some embodiments, the oligonucleotide comprises an LNA nucleoside and a 2' -O-MOE nucleoside. In some embodiments, the oligonucleotide comprises a (S) cET LNA nucleoside and a 2' -O-MOE nucleoside.

In some embodiments, the heteropolymer, or contiguous nucleotide sequence thereof, comprises only LNA and DNA nucleotides, and such LNA heteropolymer oligonucleotides can be between, for example, 8 and 24 nucleotides in length (see, e.g., WO2007112754, which discloses LNA antmiR inhibitors of micrornas).

A variety of heteropolymer compounds are very effective as therapeutic oligomers, particularly when targeting micrornas (antimirrs) or as Splice Switching Oligomers (SSOs).

In some embodiments, the hybrid comprises the following motifs

… [ L ] mDnLmDnLm … or

… [ L ] mDnLnnLnLm … or

… [ L ] mDnLmDnLnLnLm … or

…[L]m[D]n[L]m[D]n[L]m[D]n[L]m[D]n[L]m[D]n[L]m…

Wherein L represents a sugar modified nucleoside such as LNA or a 2 'substituted nucleoside (e.g. 2' -O-MOE), D represents a DNA nucleoside, and wherein each m is independently selected from 1-6, and each n is independently selected from 1,2, 3 and 4 such as 1-3. In some embodiments, each L is an LNA nucleoside. In some embodiments, at least one L is an LNA nucleoside and at least one L is a 2' -O-MOE nucleoside. In some embodiments, each L is independently selected from: LNA and 2' -O-MOE nucleosides.

In some embodiments, the heteropolymer may comprise or consist of a contiguous nucleotide sequence of between 10 and 24 nucleotides, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

In some embodiments, the contiguous nucleotide sequence of the hybrid comprises at least 30%, such as at least 40%, such as at least 50%, LNA units.

In some embodiments, the heteropolymer comprises or consists of a contiguous nucleotide sequence of nucleotide analogs and naturally occurring nucleotide repeat patterns or one type of nucleotide analog and a second type of nucleotide analog. The repeating pattern may be, for example: each second or each third nucleotide is a nucleotide analogue such as LNA and the remaining nucleotides are naturally occurring nucleotides such as DNA, or may be a 2' substituted nucleotide analogue, such as a 2' MOE of a 2' fluoro analogue as referred to herein, or in some embodiments, selected from the group of nucleotide analogues referred to herein. It will be appreciated that a repeating pattern of nucleotide analogues such as LNA units may be combined with nucleotide analogues at fixed positions (e.g. at the 5 'or 3' end).

In some embodiments, the first nucleotide of the oligomer, counted from the 3 'end, is a nucleotide analog, such as a LNA nucleotide or a 2' -O-MOE nucleoside.

In some embodiments, which may be the same or different, the second nucleotide of the oligomer, counted from the 3 'end, is a nucleotide analog, such as a LNA nucleotide or a 2' -O-MOE nucleoside.

In some embodiments, which may be the same or different, the 5 'terminus of the oligomer is a nucleotide analog, such as an LNA nucleotide or a 2' -O-MOE nucleoside.

In some embodiments, the hybrid polymer comprises at least one region comprising at least two consecutive nucleotide analogue units, such as at least two consecutive LNA units.

In some embodiments, the hybrid comprises at least one region comprising at least three consecutive nucleotide analogue units, such as at least three consecutive LNA units.

Conjugates

The term "conjugate" as used herein refers to an oligonucleotide covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

Conjugation of the oligonucleotides of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugate modulates or enhances the pharmacokinetic properties of the oligonucleotide in part by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular, the conjugates can target oligonucleotides to a particular organ, tissue, or cell type, and thereby enhance the effectiveness of the oligonucleotides in such organ, tissue, or cell type. At the same time, the conjugate can reduce the activity of the oligonucleotide in a non-target cell type, tissue or organ, e.g., off-target activity or activity in a non-target cell type, tissue or organ.

Suitable conjugate moieties are provided in WO 93/07883 and WO 2013/033230, both of which are hereby incorporated by reference. Other suitable conjugate moieties are those capable of binding to asialoglycoprotein receptor (ASGPR). In particular, trivalent N-acetylgalactosamine conjugate moieties are suitable for use in binding to ASGPR, see, e.g., WO 2014/076196, WO 2014/207232, and WO 2014/179620 (incorporated herein by reference). Such conjugates are useful for enhancing uptake of the oligonucleotide by the liver while reducing the presence of the oligonucleotide in the kidney, thereby increasing the liver/kidney ratio of the conjugated oligonucleotide compared to an unconjugated form of the same oligonucleotide.

Oligonucleotide conjugates and their synthesis have also been reported in the Manohara's comprehensive review (Antisense Drug Technology, Principles, Strategies, and Applications, S.T.) crook, ed., Ch.16, Marcel Dekker, Inc.,2001, and Manohara's Antisense and Nucleic Acid Drug Development,2002,12,103, each of which is incorporated herein by reference in its entirety.

In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate, a cell surface receptor ligand, a drug, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

Joint

A bond or linker is a connection between two atoms that links one target chemical group or segment to another target chemical group or segment via one or more covalent bonds. The conjugate moiety may be attached to the oligonucleotide directly or through a linking moiety (e.g., a linker or tether). The linker may covalently link the third region, e.g., a conjugate moiety (region C), to the first region, e.g., an oligonucleotide or a contiguous nucleotide sequence (region a) complementary to the target nucleic acid.

In some embodiments of the invention, a conjugate or oligonucleotide conjugate of the invention may optionally comprise a linker region (second region or region B and/or region Y) between the oligonucleotide or contiguous nucleotide sequence (region a or first region) complementary to the target nucleic acid and the conjugate moiety (region C or third region).

Region B refers to a biocleavable linker comprising or consisting of a physiologically labile bond that is cleavable under conditions typically encountered in the mammalian body or similar thereto. Conditions under which the physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions, such as pH, temperature, oxidizing or reducing conditions or agents, and salt concentrations encountered in, or similar to, mammalian cells. Mammalian intracellular conditions also include enzymatic activities typically present in mammalian cells, such as enzymatic activities from proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biologically cleavable linker is susceptible to cleavage by S1 nuclease. In preferred embodiments, the nuclease susceptible linker comprises between 1 and 10 nucleosides, such as 1,2, 3, 4,5, 6, 7, 8, 9 or 10 nucleosides, more preferably 2 to 6 nucleosides, most preferably 2 to 4 linked nucleosides comprising at least two consecutive phosphodiester linkages, such as at least 3 or 4 or 5 consecutive phosphodiester linkages. Preferably, the nucleoside is DNA or RNA. See WO 2014/076195 (incorporated herein by reference) for a detailed description of phosphodiesters comprising a biocleavable linker.

Region Y means a linker that is not necessarily of the biologically cleavable type but which has the primary function of covalently linking the conjugate moiety (region C or the third region) to the oligonucleotide (region a or the first region). The region Y linker may comprise a chain structure or oligomer of repeating units such as ethylene glycol units, amino acid units, or aminoalkyl groups. The oligonucleotide conjugates of the present invention may be composed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments, the linker (region Y) is an aminoalkyl group such as C2-C36 aminoalkyl groups, including, for example, C6 to C12 aminoalkyl groups. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

The invention therefore relates in particular to:

the oligonucleotide according to the invention, wherein (A)¹) And (A)²) One is a sugar-modified nucleoside and the other is DNA;

the oligonucleotide according to the invention, wherein (A)¹) And (A)²) And are sugar-modified nucleosides;

an oligonucleotide according to the invention, wherein the sugar modified nucleoside is independently a 2' sugar modified nucleoside;

the oligonucleotide according to the invention, wherein the 2 'sugar modified nucleoside is independently selected from 2' -alkoxy-RNA, in particular 2 '-methoxy-RNA, 2' -alkoxyalkoxy-RNA, in particular 2 '-methoxyethoxy-RNA, 2' -amino-DNA, 2 '-fluoro-RNA or 2' -fluoro-ANA;

the oligonucleotide according to the invention, wherein the 2' sugar-modified nucleoside is a 2' -alkoxyalkoxy-RNA, in particular a 2' -methoxyethoxy-RNA;

an oligonucleotide according to the invention, wherein the 2' sugar modified nucleoside is a LNA nucleoside;

an oligonucleotide according to the invention, wherein the LNA nucleosides are independently selected from β -D-oxy LNA, 6' -methyl- β -D-oxy LNA and ENA, in particular β -D-oxy LNA;

an oligonucleotide according to the invention, comprising a further internucleoside linkage selected from a phosphodiester internucleoside linkage, a phosphorothioate internucleoside linkage and an internucleoside linkage as defined in formula (I);

an oligonucleotide according to the invention, comprising a further internucleoside linkage selected from a phosphorothioate internucleoside linkage and an internucleoside linkage as defined in formula (I);

an oligonucleotide according to the invention, comprising between 1 and 15, in particular between 1 and 5, more in particular 1,2, 3, 4 or 5 dinucleosides of formula (I) as defined in formula (I);

the oligonucleotides according to the invention, wherein the further internucleoside linkages are all of the formula-P (═ S) (OR) O₂The phosphorothioate internucleoside linkage of (a), wherein R is hydrogen or a phosphate protecting group;

the oligonucleotide according to the invention, further comprising an additional nucleoside selected from the group consisting of DNA nucleosides, RNA nucleosides, and sugar-modified nucleosides;

an oligonucleotide according to the invention, wherein one or more nucleosides are nucleobase modified nucleosides, such as nucleosides comprising a 5-methylcytosine nucleobase;

the oligonucleotides according to the invention, wherein at least one dinucleotide of formula (I) is in the flanking region of the gapmer antisense oligonucleotide or between the gapped region and the flanking region of the gapmer antisense oligonucleotide, i.e. (A)¹) And (A)²) Both are sugar-modified nucleosides, or (A)¹) And (A)²) One is a DNA nucleoside or an RNA nucleoside and the other is a sugar-modified nucleoside;

the oligonucleotide according to the invention, wherein the gapmer oligonucleotide is an LNA gapmer, a mixed wing gapmer or a 2 '-substituted gapmer, in particular a 2' -O-methoxyethyl gapmer;

the oligonucleotide according to the invention, wherein A is sulfur.

An oligonucleotide according to the invention, wherein the antisense gapmer oligonucleotide comprises a contiguous nucleotide sequence of the formula 5' -F-G-F ' -3', wherein G is a region of 5 to 18 nucleotides capable of recruiting RNaseH, and said region G flanked at 5' and 3' by flanking regions F and F ', respectively, wherein regions F and F ' independently comprise or consist of 1 to 72 ' -sugar modified nucleotides, wherein the nucleotides of region F adjacent to region G are 2' -sugar modified nucleotides, and wherein the nucleotides of region F ' adjacent to region G are 2' -sugar modified nucleotides;

an oligonucleotide according to the invention, wherein said at least one dinucleotide of formula (I) is located in region F or F ', or between region G and region F';

an oligonucleotide according to the invention, wherein the 2 '-sugar modified nucleoside in the F region or the F' region or in both the F region and the F 'region is independently selected from the group consisting of 2' -alkoxy-RNA, in particular 2 '-methoxy-RNA, 2' -alkoxyalkoxy-RNA, in particular 2 '-methoxyethoxy-RNA, 2' -amino-DNA, 2 '-fluoro-RNA, 2' -fluoro-ANA and LNA nucleosides;

an oligonucleotide according to the invention, wherein all 2' -sugar modified nucleosides in the F region or the F ' region or both the F region and the F ' region are LNA nucleosides;

an oligonucleotide according to the invention, wherein the 2 '-sugar modified nucleosides in the F region or the F' region or in both the F region and the F 'region are both 2' -alkoxy-RNA, in particular 2 '-methoxy-RNA, both 2' -alkoxyalkoxy-RNA, in particular 2 '-methoxyethoxy-RNA, both 2' -amino-DNA, both 2 '-fluoro-RNA, both 2' -fluoro-ANA or both LNA nucleosides;

an oligonucleotide according to the invention, wherein the F region or the F 'region or both the F region and the F' region comprise at least one LNA nucleoside and at least one DNA nucleoside;

an oligonucleotide according to the invention, wherein the F region or the F 'region or both the F region and the F' region comprise at least one LNA nucleoside and at least one non-LNA 2 '-sugar modified nucleoside, such as at least one 2' -methoxyethoxy-RNA nucleoside;

the oligonucleotide according to the invention, wherein the gap region G comprises 5 to 16, particularly 8 to 16, more particularly 8, 9,10,11, 12, 13 or 14 consecutive DNA nucleosides;

an oligonucleotide according to the invention, wherein region F and region F' are independently 1,2, 3, 4,5, 6, 7 or 8 nucleosides in length;

an oligonucleotide according to the invention, wherein region F and region F' each independently comprise 1,2, 3 or 4 LNA nucleosides;

an oligonucleotide according to the invention, wherein the LNA nucleosides are independently selected from β -D-oxy LNA, 6' -methyl- β -D-oxy LNA and ENA;

an oligonucleotide according to the invention, wherein the LNA nucleoside is β -D-oxolna;

an oligonucleotide according to the invention, wherein the oligonucleotide or a contiguous nucleotide sequence thereof (F-G-F') is 10 to 30 nucleotides in length, in particular 12 to 22, more in particular 14 to 20 oligonucleotides in length;

the oligonucleotide according to the invention, wherein the gapmer oligonucleotide comprises a contiguous nucleotide sequence of the formula 5'-D' -F-G-F '-D "-3', wherein F, G and F 'are as defined in any of claims 17 to 28, wherein the regions D' and D" each independently consist of 0 to 5 nucleotides, in particular 2, 3 or 4 nucleotides, in particular DNA nucleotides (such as phosphodiester linked DNA nucleosides);

the oligonucleotide according to any one of claims 17 to 29, wherein each flanking region F and F' independently comprises 1,2, 3, 4,5, 6 or 7, in particular one dinucleotide of formula (I);

the oligonucleotides according to the invention comprise in total one dinucleotide of the formula (I), in particular one dinucleotide of the formula (I) in the region F 'or between the region G and the region F'.

An oligonucleotide according to the invention, wherein the oligonucleotide is capable of recruiting human RNaseH 1;

pharmaceutically acceptable salts of the oligonucleotides according to the invention, in particular the sodium, potassium or ammonium salts;

a conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt according to the invention and at least one conjugate moiety covalently attached to the oligonucleotide or the pharmaceutically acceptable salt, optionally via a linker moiety;

a pharmaceutical composition comprising an oligonucleotide, a pharmaceutically acceptable salt or conjugate according to the invention and a therapeutically inert carrier;

the oligonucleotide, pharmaceutically acceptable salt or conjugate according to the invention for use as therapeutically active substance.

The invention relates in particular to a compound of formula (I-a)

Wherein

R²Is alkoxy, alkoxyalkoxy or amino; and is

R⁴Is hydrogen; or

R⁴And R²Together form X-Y;

x is oxygen, sulfur or-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(＝CR^aR^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-；-O-NR^a-、-NR^a-O-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

Y is oxygen, sulfur, - (CR)^aR^b)_n-、-CR^aR^b-O-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

Conditionis-X-Y-is not-O-, Si (R)^a)₂-Si(R^a)₂-、-SO₂-SO₂-、-C(R^a)＝C(R^b)-C(R^a)＝C(R^b)、-C(R^a)＝N-C(R^a)＝N-、-C(R^a)＝N-C(R^a)＝C(R^b)、-C(R^a)＝C(R^b)-C(R^a) N-or-Se-;

j is oxygen, sulfur, ═ CH₂Or ═ N (R)^a)；

R^aAnd R^bIndependently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, mercapto, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, ureido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, mercaptoalkylthio, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC (═ X^a)R^c、-OC(＝X^a)NR^cR^dand-NR^eC(＝X^a)NR^cR^d；

Or two geminal R^aAnd R^bTogether form an optionally substituted methylene group;

or two geminal R^aAnd R^bTogether with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl group, and only one carbon atom in-X-Y-has this condition;

wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy, and substituted methylene are alkyl, alkenyl, alkynyl, and methylene substituted with 1 to 3 substituents independently selected from the group consisting of: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl and heteroaryl;

X^ais oxygen, sulfur or-NR^c；

R^c、R^dAnd R^eIndependently selected from hydrogen and alkyl;

R⁵is a hydroxyl protecting group;

R^xis cyanoalkyl or alkyl;

R^yis dialkylamino or pyrrolidinyl;

nu is a nucleobase or a protected nucleobase; and is

n is 1,2 or 3.

The oligonucleotides according to the invention can be prepared, for example, according to the following scheme.

Scheme 2

In scheme 2, B1 and B2 are nucleobases, and a is as defined above.

Oligonucleotides comprising phosphonoacetate or thiophosphonoacetate modifications can be chemically synthesized using solid phase oligonucleotides. DMT-protected deoxyribonucleoside 3' -O-diisopropylaminophosphino acetic acid dimethyl-beta-cyanoethyl ester is condensed to a solid support-attached deoxyribonucleoside. Followed by the use of, for example, a suboxidant reagent (0.02M I)₂THF/pyridine/H of₂Solution O: 88/10/2) oxidizing or sulfurizing the phosphonite bond using, for example, a 0.1M solution of 3-amino-1, 2, 4-dithiazole-5-thione in acetonitrile/pyridine. After capping with acetic anhydride and treatment with dichloroacetic acid to remove the 5' -O-dimethoxytriyl group, the cycle is repeated an appropriate number of times to provide an oligonucleotide containing a phosphonoacetate modification.

The monomer building blocks that can be used for the manufacture of the oligonucleotides according to the invention can be prepared, for example, according to the following scheme.

Dimethyl cyanoethyl bromoacetate was synthesized by condensing bromoacetyl bromide with 3-hydroxy-3-methylbutyronitrile in toluene at reflux overnight. The phosphite derivative is then prepared via a Reformatsky reaction with diisopropylaminophosphine. This reactant is further condensed with a protected 2' -deoxynucleoside using tetrazole to give the LNA PACE phosphoramidite.

Scheme 3

In scheme 3, R⁵、R^x、R^yAnd Nu is as defined above.

The monomers can be prepared according to the following scheme, in particular following the above procedure.

Scheme 4

In scheme 4, Nu is as defined above.

Accordingly, the present invention relates to a compound of formula (II)

Wherein

X is oxygen, sulfur or-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(＝CR^aR^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-；-O-NR^a-、-NR^a-O-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

Y is oxygen, sulfur, - (CR)^aR^b)_n-、-CR^aR^b-O-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

With the proviso that-X-Y-is not-O-O-, Si (R)^a)₂-Si(R^a)₂-、-SO₂-SO₂-、-C(R^a)＝C(R^b)-C(R^a)＝C(R^b)、-C(R^a)＝N-C(R^a)＝N-、-C(R^a)＝N-C(R^a)＝C(R^b)、-C(R^a)＝C(R^b)-C(R^a) N-or-Se-;

j is oxygen, sulfur, ═ CH₂Or ═ N (R)^a)；

R^aAnd R^bIndependently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, mercapto, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, ureido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, mercaptoalkylthio, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC (═ X^a)R^c、-OC(＝X^a)NR^cR^dand-NR^eC(＝X^a)NR^cR^d；

Or two geminal R^aAnd R^bTogether form an optionally substituted methylene group;

or two geminal R^aAnd R^bTogether with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl group, and only one carbon atom in-X-Y-has this condition;

wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy, and substituted methylene are alkyl, alkenyl, alkynyl, and methylene substituted with 1 to 3 substituents independently selected from the group consisting of: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl and heteroaryl;

X^ais oxygen, sulfur or-NR^c；

R^c、R^dAnd R^eIndependently selected from hydrogen and alkyl;

R⁵is a hydroxyl protecting group;

R^xis cyanoalkyl or alkyl, especially cyanoalkyl;

R^yis dialkylamino or pyrrolidinyl; and is

Nu is a nucleobase or a protected nucleobase; and is

n is 1,2 or 3;

or a pharmaceutically acceptable salt thereof.

The invention also relates in particular to:

the compound according to the present invention, wherein-X-Y-is-CH₂-O-、-CH(CH₃) -O-or-CH₂CH₂-O-；

The compound according to the invention, which has the formula (III) or (IV)

Wherein R is⁵、R^x、R^yAnd Nu is as defined above;

the compound according to the invention, wherein R^xIs 2-cyano-1, 1-dimethyl-ethyl, methyl, ethyl, propyl or tert-butyl;

the compound according to the invention, wherein R^xIs 2-cyano-1, 1-dimethyl-ethyl;

the compound according to the invention, wherein R^yIs diisopropylamino or pyrrolidinyl;

the compound according to the invention, wherein R^yIs a dialkylamino group;

a compound according to any one of claims 1 to 6, wherein R^yIs diisopropylamino.

The compounds according to the invention are of the formula (V)

Wherein R is⁵And Nu is as defined above;

the compound according to the present invention, wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil or protected uracil.

The compounds according to the invention are selected from

And

a process for the manufacture of a compound of formula (II) according to the invention, which comprises reacting a compound of formula (C)

And formula P (R)^y)₂(CH₂)COO(R^x) In the presence of a coupling agent and a base, wherein X, Y, R⁵、Nu、R^xAnd R^yAs defined above;

the method according to the invention, wherein the coupling agent is 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole or 4, 5-Dicyanoimidazole (DCI), in particular tetrazole; and is

Use of a compound according to the invention in the manufacture of an oligonucleotide.

The process of the invention may conveniently be quenched with a base, for example with triethylamine, pyridine, diisopropylamine or N, N-diisopropylethylamine.

Oligonucleotides according to the invention comprising 2 '-alkoxy-RNA, in particular 2' -methoxy-RNA, 2 '-alkoxyalkoxy-RNA, in particular 2' -methoxyethoxy-RNA, can be synthesized according to the following procedure.

Scheme 5

In scheme 5, B1 and B2 are nucleobases, and a is as defined above.

Oligonucleotides comprising MOE (or other 2' substituent) phosphonoacetate or thiophosphonoacetate modifications can be chemically synthesized using solid phase oligonucleotides. DMT-protected deoxyribonucleoside 3' -O-diisopropylaminophosphino acetic acid dimethyl-beta-cyanoethyl ester is condensed to a solid support-attached deoxyribonucleoside. Followed by the use of, for example, a suboxidant reagent (0.02M I)₂THF/pyridine/H of₂Solution O: 88/10/2) oxidizing or sulfurizing the phosphonite bond using, for example, a 0.1M acetonitrile/pyridine solution of 3-amino-1, 2, 4-dithiazole-5-thione. After capping with acetic anhydride and treatment with dichloroacetic acid to remove the 5' -O-dimethoxytriyl group, the cycle is repeated an appropriate number of times to provide an oligonucleotide containing a phosphonoacetate modification.

The monomer building blocks that can be used for the manufacture of the oligonucleotides according to the invention can be prepared, for example, according to the following scheme.

Dimethyl cyanoethyl bromoacetate was synthesized by condensing bromoacetyl bromide with 3-hydroxy-3-methylbutyronitrile in toluene at reflux overnight. The phosphite derivative is then prepared via a Reformatsky reaction with diisopropylaminophosphine. This reactant was further condensed with a protected 2' -deoxynucleoside using 4,5-DCI to give a MOE PACE phosphoramidite.

Scheme 6

In scheme 6, R⁵、R^x、R^yAnd Nu is as defined above.

The monomers can be prepared according to the following scheme, in particular following the above procedure.

Scheme 7

In scheme 7, Nu is as defined above.

Accordingly, the present invention relates to a compound of formula (VI)

Wherein

R²Is alkoxy, alkoxyalkoxy or amino, in particular alkoxy or alkoxyalkoxy;

R⁵is a hydroxyl protecting group;

R^xis cyanoalkyl or alkyl, especially cyanoalkyl;

R^yis dialkylamino or pyrrolidinyl; and is

Nu is a nucleobase or a protected nucleobase; and is

Or a pharmaceutically acceptable salt thereof.

The invention also relates in particular to:

the compound according to the invention, wherein R²Is methoxy, methoxyethoxy or amino, in particular methoxy or methoxyethoxy;

the compounds according to the invention are of the formula (VII)

Wherein R is⁵、R^x、R^yAnd Nu is as defined above;

the compound according to the invention, wherein R^xIs 2-cyano-1, 1-dimethyl-ethyl, methyl, ethyl, propyl or tert-butyl;

the compound according to the invention, wherein R^xIs 2-cyano-1, 1-dimethyl-ethyl;

the compound according to the invention, wherein R^yIs diisopropylamino or pyrrolidinyl;

the compound according to the invention, wherein R^yIs a dialkylamino group;

a compound according to any one of claims 1 to 6, wherein R^yIs diisopropylamino.

The compounds according to the invention are of the formula (VIII)

Wherein R is⁵And Nu is as defined above;

the compound according to the present invention, wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil or protected uracil.

The compounds according to the invention are selected from

And

a process for the manufacture of a compound of formula (VI) according to the invention, which comprises reacting a compound of formula (D)

And formula P (R)^y)₂(CH₂)COO(R^x) In the presence of a coupling agent and a base, wherein R²、R⁵、Nu、R^xAnd R^yAs defined above;

the method according to the invention, wherein the coupling agent is 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4, 5-Dicyanoimidazole (DCI), in particular DCI; and is

Use of a compound according to the invention in the manufacture of an oligonucleotide.

The process of the invention may conveniently be quenched with a base, for example with triethylamine, pyridine, diisopropylamine or N, N-diisopropylethylamine.

The invention will now be illustrated by the following examples, which are not limiting.

Examples of the invention

Abbreviations:

a adenine

G-guanine

_mC methyl cytosine

T-Thymidine

LNA locked nucleic acids

RNA ribonucleic acid

DMT dimethoxytrityl radical

DCA Dichloroacetic acid

DCM dichloromethane

THF tetrahydrofuran

Anh. anhydrous

TLC thin layer chromatography

NMR nuclear magnetic resonance

CPG (compact peripheral component interconnect) controllable aperture glass

Reverse transcription of RT

qPCR quantitative polymerase chain reaction

ds double strand

Tm Hot melting Point

Example 1: monomer synthesis

1.1.1-cyano-2-methylpropan-2-yl 2-bromoacetate

To a solution of 2-bromoacetyl bromide (14.7g, 6.31mL, 72.6mmol, 1.2 equiv.) in toluene (67.2mL) was slowly added 3-hydroxy-3-methylbutyronitrile (6g, 6.28mL, 60.5mmol, 1 equiv.) with stirring. The round bottom flask was fitted with a Friedrich condenser and a drying tube to an acid separator (containing aqueous NaOH). The reaction mixture was heated to reflux overnight. The reaction was allowed to cool to room temperature and the compound was then concentrated in vacuo to give a colorless oil. The crude product was purified by Combiflash chromatography using ethyl acetate/hexane as gradient, eluting the product in 30% ethyl acetate in hexane to give 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (8.14g, 37mmol, 58% yield).¹H NMR(CHLOROFORM-d,300MHz)δ3.8(s,2H),2.9(s,2H),1.6(s,6H)。

1.2.1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate

1-chloro-N, N, N ', N' -tetraisopropylphosphinediamine (7.75g, 29mmol, 1 eq.) was dissolved in anhydrous THF (69.4 ml). An additional 41.6ml of anhydrous diethyl ether was added. A solution of 1-cyano-2-methylpropan-2-yl 2-bromoacetate (7.03g, 32mmol, 1.1 equiv.) in anhydrous THF (34.7ml) was placed in a round bottom flask. Adding zinc (2.85g, 43.6mmol, 1.5 equiv.), anhydrousDiethyl ether (22.2mL) and a magnetic stir bar were placed in a 500mL three-necked round bottom flask fitted with a Friedrich condenser. Phosphine (36mL) and bromoacetate solution (10mL) were added simultaneously and very slowly to a three-neck round-bottom flask. The reaction mixture was then heated at reflux until an exothermic reaction was evident (slightly cloudy, colorless reaction became clear and light yellow). The reaction was continued at reflux by adding the remaining phosphine and bromoacetate solution. Once the addition was complete, the reaction was held at reflux for 45 minutes by heating, allowed to cool to room temperature and passed³¹P NMR analysis was complete. The starting material at δ -135 ppm was converted to a single product at δ -48 ppm. The cooled reaction mixture was concentrated in vacuo to give a viscous oil. The resulting material was dissolved with anhydrous heptane and a small amount of acetonitrile to fully dissolve the crude product. The solution was extracted twice with dry heptane. By passing³¹P NMR analysis of the acetonitrile layer determined that no product was present at δ 48ppm and was discarded. All heptane fractions were combined and concentrated in vacuo to give a pale yellow oil. It was then dried under high vacuum overnight to give a white solid (7.096g, 19mmol, 62% yield).¹H NMR(CHLOROFORM-d,300MHz)δ3.3-3.5(m,4H),2.9(s,2H),2.7(d,2H),1.60(s,6H),1.3(m,24H)。

1.3. (1-cyano-2-methylpropan-2-yl) 2- [ [ di (prop-2-yl) amino ] - [ [ rac- (1R,3R) -1- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -3- (5-methyl-2, 4-dioxopyrimidin-1-yl) -2, 5-dioxabicyclo [2.2.1] hept-7-yl ] oxy ] phosphono ] acetate

1- [ (1R,4R,6R,7S) -4- [ [ bis (4-methoxyphenyl) -phenyl-methoxy]Methyl radical]-7-hydroxy-2, 5-dioxabicyclo [2.2.1]Hept-6-yl]-5-methyl-pyrimidine-2, 4-dione (0.7g, 1.22mmol, 1 equiv.) was dissolved in anhydrous DCM (15.3ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (545mg, 1.47mmol, 1.2 equiv.) was added to the reaction mixture. After complete dissolution of the reaction components, tetrazole (2.17ml, 978. mu. mol, 0.8 equivalents) was added) With 0.45M anhydrous CH₃CN solution was added to the reaction mixture. The reaction mixture was then stirred under argon at room temperature overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate). By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. Upon completion, the reaction was quenched by the addition of triethylamine (99mg, 136. mu.l, 978. mu. mol, 0.8 eq.). After 5 min, the reaction mixture was concentrated in vacuo to give a viscous colorless oil. The product was redissolved in a minimum volume of ethyl acetate and purified by column chromatography (80/20: ethyl acetate/heptane). The product containing fractions were combined and concentrated to give a foam, which was redissolved in a minimum volume of anhydrous DCM. Heptane was added dropwise with rapid stirring. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 743mg of the title compound as a white solid (743mg, 0.88mmol, 69% yield).³¹P NMR(CHLOROFORM-d,121MHz)δ126.91(s,1P),122.25(s,1P)。¹H NMR (600MHz, acetonitril-d 3) δ ppm 8.89-9.22(m,1H),7.57-7.59(m,1H),7.50(d, J ═ 7.6Hz,1H),7.33-7.39(m,3H),7.33-7.37(m,2H),7.26-7.31(m,1H),6.88-6.95(m,4H),5.58(s,1H),4.62(s,1H),4.14(dJ, ═ 6.8Hz,1H),3.79-3.81(m,5H),3.79-3.85(m,2H),3.47-3.50(m,2H),3.42-3.50(m,1H),2.92-2.95(m,1H), 2.67-2.67 (m,2H), 3.71H, 6H, 1H, 5H, 7.71H, 6H, 1H, 6H, 5H, 3.60H, 5H, 4H) in that respect LCMS (ES +) found: 843.37 g/mol.

1.4. (1-cyano-2-methylprop-2-yl) 2- [ [ di (prop-2-yl) amino ] - [ [ rac- (1R,3R) -3- (6-benzamidopurin-9-yl) -1- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -2, 5-dioxabicyclo [2.2.1] hept-7-yl ] oxy ] phosphono ] acetate

Reacting N- [9- [ (1R,4R,6R,7S) -4- [ [ bis (4-methoxyphenyl) -phenyl-methoxy]Methyl radical]-7-hydroxy-2, 5-dioxabicyclo [2.2.1]Hept-6-yl]Purin-6-yl]Benzamide (3g, 4.37)mmol, 1 eq) was dissolved in anhydrous DCM (54.7ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (1.95g, 5.25mmol, 1.2 eq) was added to the reaction mixture. After complete dissolution of the reaction components, tetrazole (7.78ml, 3.5mmol, 0.8 equiv.) is reacted with 0.45M anhydrous CH₃CN solution was added to the reaction mixture. The reaction mixture was stirred at room temperature under argon overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate). By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. Upon completion, the reaction was quenched by the addition of triethylamine (354mg, 488 μ l, 3.5mmol, 0.8 eq). After 5 min, the reaction mixture was concentrated in vacuo to give a viscous colorless oil. The product was redissolved in a minimum volume of ethyl acetate and purified by column chromatography (80/20: ethyl acetate/heptane). The product containing fractions were combined and concentrated to give a foam, which was redissolved in a minimum volume of anhydrous DCM. Heptane was added dropwise with rapid stirring. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 1.86g of the title compound as a white solid (1.86g, 1.9mmol, 45% yield).³¹P NMR(ACETONITRILE-d₃121MHz) delta 125.2(s,1P),120.9(s, 1P). LCMS (ES +) found: 956.40 g/mol.

1.5. (1-cyano-2-methylpropan-2-yl) 2- [ [ di (prop-2-yl) amino ] - [ [ rac- (1R,3R) -3- (4-benzamido-5-methyl-2-oxopyrimidin-1-yl) -1- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -2, 5-dioxabicyclo [2.2.1] hept-7-yl ] oxy ] phosphono ] acetate

Reacting N- [1- [ (1R,4R,6R,7S) -4- [ [ bis (4-methoxyphenyl) -phenyl-methoxy]Methyl radical]-7-hydroxy-2, 5-dioxabicyclo [2.2.1]Hept-6-yl]-5-methyl-2-oxo-pyrimidin-4-yl]Benzamide (2.8g, 4.14mmol, 1 eq) was dissolved in anhydrous DCM (59.2ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropyl) amide was then addedPhenylamino) phosphono) acetate (1.85g, 4.97mmol, 1.2 equivalents) was added to the reaction mixture. After complete dissolution of the reaction components, tetrazole (7.37ml, 3.31mmol, 0.8 equiv.) is reacted with 0.45M anhydrous CH₃CN solution was added to the reaction mixture. The reaction mixture was stirred at room temperature under argon overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate). By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. After completion, the reaction was quenched by addition of triethylamine (335mg, 462. mu.l, 3.31mmol, 0.8 eq.). After 5 min, the reaction mixture was concentrated in vacuo to give a viscous pale yellow oil. The product was redissolved in a minimum volume of ethyl acetate and purified by column chromatography (50/50: ethyl acetate/heptane). The product containing fractions were combined and concentrated to give a foam, which was redissolved in a minimum volume of anhydrous DCM. Heptane was added dropwise with rapid stirring. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 2.35g of the title compound as a pale yellow solid (2.35g, 2.22mmol, 46% yield).³¹PNMR(ACETONITRILE-d₃121MHz) δ 126.78(s,1P),122.73(s, 1P). LCMS (ES +) found: 947.41 g/mol.

1.6. (1-cyano-2-methylpropan-2-yl) 2- [ [ di (prop-2-yl) amino ] - [ [ rac- (1R,3R) -1- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -3- [2- (2-methylpropanamino) -6-oxo-1H-purin-9-yl ] -2, 5-dioxabicyclo [2.2.1] hept-7-yl ] oxy ] phosphono ] acetate

N' - [9- [ (1R,4R,6R,7S) -4- [ [ bis (4-methoxyphenyl) -phenyl-methoxy ] group]Methyl radical]-7-hydroxy-2, 5-dioxabicyclo [2.2.1]Hept-6-yl]-6-oxo-1H-purin-2-yl]-N, N-dimethyl-formamidine (2.6g, 3.89mmol, 1 equivalent) was dissolved in anhydrous DCM (55.6ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (1.74g, 4.67mmol, 1.2 equivalents) was added to the reaction mixIn the above-mentioned material. After the reaction components were completely dissolved, tetrazole (6.92ml, 3.12mmol, eq: 0.8) was reacted with 0.45M anhydrous CH₃CN solution was added to the reaction mixture. The reaction mixture was stirred at room temperature under argon overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate). By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. Upon completion, the reaction was quenched by the addition of triethylamine (315mg, 434. mu.l, 3.12mmol, 0.8 eq.). After 5 min, the reaction mixture was concentrated in vacuo to give a viscous colorless oil. The product was redissolved in a minimum volume of ethyl acetate and purified by column chromatography (100% ethyl acetate). The product containing fractions were combined and concentrated to give a foam, which was redissolved in a minimum volume of anhydrous DCM. Heptane was added dropwise with rapid stirring. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 1.4g of the title compound as a white solid (1.4g, 1.4mmol, 38% yield).³¹P NMR(ACETONITRILE-d₃121MHz) δ 126.48(s,1P),121.3(s, 1P). LCMS (ES +) found: 938.42 g/mol.

Example 2: oligonucleotide synthesis

Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer from Bioautomation. Using controlled aperture glass supports with universal joints

The synthesis was performed on a 1. mu. mol scale.

In a standard cycling procedure for coupling standard DNA and LNA phosphoramidites, 3% (w/v) of CH in dichloroacetic acid was used₂Cl₂The solution was DMT deprotected in three applications, 230. mu.L each, for 105 seconds. 95 μ L of a 0.1M acetonitrile solution (or for LNA-^MeC building a block of acetonitrile/CH₂Cl₂1:1 solution) and 110. mu.L of 0.25M 5- [3, 5-bis (trifluoromethyl) phenyl]-2H-tetrazole as activator and a coupling time of 180 seconds was coupled three times with the corresponding phosphoramidite. Using a 0.1M acetonitrile/pyridine solution of 3-amino-1, 2, 4-dithiazole-5-thione at one timeVulcanization was carried out in the application at a volume of 200. mu.L for 3 minutes. Use 0.02M I₂THF/pyr/H of₂The solution of O88/10/2 was oxidized in one application for 3 minutes. Using THF/lutidine/Ac₂O8: 1:1(CapA, 75. mu. mol) and THF/N-methylimidazole 8:2(CapB, 75. mu. mol) were capped for 70 seconds.

The synthesis cycle for introducing PACE LNA comprises the use of CH with 3% (w/v) dichloroacetic acid₂Cl₂The solution was DMT deprotected in three applications, 230. mu.L each, for 105 seconds. With 95. mu.L of a 0.1M acetonitrile solution and 110. mu.L of a 0.25M 5- [3, 5-bis (trifluoromethyl) phenyl group]The freshly prepared LNA PACE was coupled twice with a coupling time of 15 minutes using-2H-tetrazole solution as activator. Sulfurization was carried out using 0.1M 3-amino-1, 2, 4-dithiazole-5-thione in acetonitrile/pyridine solution in one application for 3 minutes. Use 0.02M I₂THF/pyr/H of₂The solution of O88/10/2 was oxidized in one application for 3 minutes. Using THF/lutidine/Ac₂O8: 1:1(CapA, 75. mu. mol) and THF/N-methylimidazole 8:2(CapB, 75. mu. mol) were capped for 70 seconds.

After synthesis, 1.5% DBU was in anhydrous CH₃The solution in CN was carefully passed through the column several times to deprotect the dimethylcyanoethyl protecting group and prevent alkylation of the base during deprotection. Then allowed to stand at room temperature for 60 minutes. The solution was then discarded and 2-3mL of anhydrous CH was used₃CN washes the column. It was then dried under a stream of argon. The CPG was then carefully transferred to a 4mL vial to which was added 1mL of 7N NH₃MeOH and stirred at 55 ℃ for 24 hours.

Crude DMT-on oligonucleotides were purified by RP-HPLC purification using a C18 column followed by DMT removal by precipitation with 80% aqueous acetic acid and ethanol or by column purification. PACE LNA phosphoramidites are synthesized in basel. Normal phosphoramidite and all reagents for solid phase synthesis were ordered from Sigma Aldrich.

The following molecules have been prepared according to the procedure described above.

PACE phosphorothioate modifications between adjacent nucleotides

A、G、^mC. T represents LNA nucleotide

a. g, c, t represent DNA nucleotides

All other bonds being prepared as phosphorothioates

Example 3: in vitro efficacy of an oligonucleotide targeting HIF1a mRNA in human HeLa and A549 cells at different concentrations of the dose response Curve

HeLa and A549 cell lines were purchased from ATCC and maintained at 37 ℃ and 5% CO according to supplier's recommendations₂In a humidified incubator. For the assay, cells were seeded at 3000 per well (HeLa) and 3500 per well (a549) in medium in 96-well plates. Cells were cultured for 24 hours, and then oligonucleotides dissolved in PBS were added. Concentration range of oligonucleotide: the maximum concentration is 25. mu.M, and 1:1 dilution is carried out in 8 steps. Three days after addition of the oligonucleotides, cells were harvested. RNA was extracted using the PureLink Pro 96RNA purification kit (Thermo Fisher Scientific) and eluted in 50. mu.l of water according to the manufacturer's instructions. The RNA was then diluted 10-fold with DNase/RNase free water (Gibco) and heated to 90 ℃ for one minute.

For gene expression analysis, qScript was used^TMXLT One-Step RT-qPCR

Low ROX^TM(Quanntadio) one-step RT-qPCR was performed in a duplex setting. The following TaqMan primer assays were used for qPCR: HIF1A, Hs00936368_ m1 and endogenous controls GUSB, Hs99999908_ m1 (VIC-MGB). All primer sets were purchased from Thermo Fisher Scientific. Relative expression levels of HIF1A mRNA were shown as a percentage of control (PBS-treated cells), and IC has been determined using GraphPad Prism7 from data for n-2 biological replicates₅₀The value is obtained.

The results are shown in the following table and in fig. 1.

The data depicted in fig. 1 are reported in the following table.

HIF1A expression in HeLa (average of biological replicates)

	#1	#2	#3	#4	#5	#6	Reference to
								25,00μM	16	17	13	25	16	20	16
12,50μM	23	26	20	39	24	27	23
								6,25μM	36	42	28	55	37	43	34
3,13μM	55	66	41	69	52	58	52
								1,56μM	70	78	61	80	72	64	66
0,78μM	78	77	76	84	76	79	74
								0,39μM	83	95	82	90	85	94	81
0,20μM	91	92	84	88	103	91	84

HIF1A expression in A549 (average of biological replicates)

Example 4: in vitro potency and efficacy of oligonucleotides targeting MALAT1 mRNA in human HeLa and A549 cells at different concentrations of the dose-response curve

HeLa and A549 cellsPurchased from ATCC and maintained at 37 ℃ and 5% CO according to supplier's recommendations₂In a humidified incubator. For the assay, cells were seeded at 3000 per well (HeLa) and 3500 per well (a549) in medium in 96-well plates. Cells were cultured for 24 hours, and then oligonucleotides dissolved in PBS were added. Concentration range of oligonucleotide: the maximum concentration is 25. mu.M, and 1:1 dilution is carried out in 8 steps. Three days after addition of the oligonucleotides, cells were harvested. RNA was extracted using the PureLink Pro 96RNA purification kit (Thermo Fisher Scientific) and eluted in 50. mu.l of water according to the manufacturer's instructions. The RNA was then diluted 10-fold with DNase/RNase free water (Gibco) and heated to 90 ℃ for one minute.

For gene expression analysis, qScript was used^TMXLT One-Step RT-qPCR

Low ROX^TM(Quanntadio) one-step RT-qPCR was performed in a duplex setting. The following TaqMan primer assays were used for qPCR: MALAT1, Hs00273907_ s1(FAM-MGB) and the endogenous control GAPDH. All primer sets were purchased from Thermo Fisher Scientific. Relative expression levels of MALAT1 mRNA were shown as a percentage of control (PBS-treated cells), and IC has been determined using GraphPad Prism7 from data for n-2 biological replicates₅₀The value is obtained.

The results are shown in the following table and in fig. 2.

The data depicted in fig. 2 is reported in the following table.

MALAT1 expression in HeLa (average of biological replicates):

	#7	#8	#9	#10	#11	#12	reference to
								25,00μM	5	4	3	3	3	3	6
12,50μM	6	5	4	3	3	4	7
								6,25μM	9	7	7	5	4	5	9
3,13μM	13	13	8	7	7	8	15
								1,56μM	23	22	14	10	12	13	22
0,78μM	29	27	32	19	19	20	37
								0,39μM	49	40	35	32	40	37	64
0,20μM	73	65	77	64	67	70	79

MALAT1 expression (average of biological replicates) in A549HeLa

	#7	#8	#9	#10	#11	#12	Reference to
								25,00μM	8	7	5	5	5	4	12
12,50μM	9	9	7	7	6	6	14
								6,25μM	13	11	11	10	10	9	18
3,13μM	22	18	18	14	14	13	27
								1,56μM	31	32	30	25	24	22	38
0,78μM	45	44	43	35	38	37	51
								0,39μM	64	66	67	56	57	50	71
0,20μM	80	86	90	79	76	79	96

Example 5: in vitro potency and efficacy of oligonucleotides targeting ApoB mRNA in mouse primary hepatocytes

Primary mouse hepatocytes were isolated from the liver of C57BL/6J mice anesthetized with pentobarbital following a 2-step perfusion protocol according to the literature (Berry and Friend,1969, J.cell Biol; Paterna et al, 1998, Toxicol.Appl.Pharmacol.). The first step was performed with HBSS +15mM HEPES +0.4mM EGTA for 5 minutes followed by HBSS +20mM NaHCO 3+ 0.04% BSA (Sigma # A7979) +4mM CaCl 2(Sigma #21115) +0,2mg/ml collagenase type 2 (Worthington #4176) for 12 minutes. Hepatocytes were captured in 5ml of cold Williams medium e (wme) (Sigma # W1878, supplemented with 1x Pen/Strep/glutamine, 10% (v/v) FBS (ATCC #30-2030)) on ice. The crude cell suspension was filtered through 70 μ M and 40 μ M cell filters (Falcon #352350 and #352340) followed by filling to 25ml with WME and centrifugation at 50x g for 5 min at room temperature to pellet hepatocytes. The supernatant was removed and the hepatocytes were resuspended in 25ml WME. After addition of 25ml of 90% Percoll solution (Sigma # P4937; pH 8.5-9.5) and centrifugation at 50x g for 10 min at 25 ℃, the supernatant and floating cells were removed. To remove the remaining Percoll, the pellet was resuspended in 50mL WME medium, centrifuged at 50x g for 3 minutes at 25 ℃, and the supernatant was discarded. The cell pellet was resuspended in 20mL WME, cell number and viability determined (Invitrogen, Cellcount) and diluted to 250,000 cells/mL. These were seeded at 25,000 cells/well in Collagen-coated 96-well plates (PD Biocoat Collagen I #356407) and cultured at 37 ℃ under 5% CO 2. After 3 hours, cells were washed with WME to remove non-attached cells, and the medium was changed. 24 hours after inoculation, oligonucleotides were added at a range of concentrations: the maximum concentration was 3,125. mu.M, and the semilog dilution was carried out in 8 steps. Three days after addition of the oligonucleotides, cells were harvested. RNA was extracted using the PureLink Pro 96RNA purification kit (Thermo Fisher Scientific) and eluted in 50. mu.l of water according to the manufacturer's instructions. The RNA was then diluted 10-fold with DNase/RNase free water (Gibco) and heated to 90 ℃ for one minute.

For gene expression analysis, qScript was used^TMXLT One-Step RT-qPCR

Low ROX^TM(Quanntadio) one-step RT-qPCR was performed in a duplex setting. The following TaqMan primer assays were used for qPCR: apob Mm-01545150-m 1(FAM-MGB) with endogenous pairsAccording to Gapdh, Mm99999915_ g1 (VIC-MGB). All primer sets were purchased from Thermo Fisher Scientific. The relative expression level of ApoB mRNA is shown as a percentage of control (PBS treated cells) and IC50 values have been determined using GraphPad Prism 7.

The results are shown in the following table and in fig. 3.

The data depicted in fig. 3 is reported in the following table.

Relative expression of ApoB mRNA in mouse primary hepatocytes

	#13	#14	#15	#16	#17	#18	#19	#20	#21	#22	#23	Reference to
													3,125μM	13	11	12	13	12	16	13	11	11	11	18	16
0,989μM	12	13	14	13	18	22	20	13	14	14	24	15
													0,313μM	16	19	22	19	27	30	28	20	24	26	42	26
0,099μM	25	42	44	41	59	56	43	42	40	33	62	34
													0,031μM	54	73	72	75	79	86	81	67	60	76	75	44
0,010μM	73	81	86	83	89	88	86	74	113	127	89	69
													0,003μM	94	87	92	86	86	86	85	104	108	89	83	88
0,001μM	94	108	110	117	120	111	102	96	89	83	91	88

Example 6: thermal melting Point (Tm) of oligonucleotides containing nucleoside Phosphonoacetate linkages that hybridize to RNA and DNA

The denaturation point (thermal melting point Tm) of dsLNA/DNA or dsLNA/RNA heteroduplexes was measured according to the following procedure:

solutions of equimolar amounts of RNA or DNA and LNA oligonucleotides (20. mu.M for ApoB and 10. mu.M for Malat-1) in buffer (137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄pH 7.4) to yield 10. mu.M dsOligonucleotides (ApoB) and 5. mu.M dsOligonucleotides (Malat-1). The solution was heated to 95 ℃ for 2 minutes (hybridization), and then allowed to cool to room temperature for 15 minutes. UV absorbance at 260nm was recorded using an Evolution 600UV-Vis spectrophotometer from Thermo Scientific (heating rate 1 ℃ per minute; reading rate twenty times per minute). To determine the denaturation point (i.e. melting point, Tm), the melting transition is fitted to a LOWESS curve, the inflection point (Tm) being determined by the peak position of the first derivative of the descriptive fit.

The Tm measurements (RNA and DNA) of the ApoB oligonucleotides are shown in the table below.

The compounds according to the invention maintain high affinity for control RNA and DNA.

Example 7: in vitro potency and efficacy of selected oligonucleotides targeting MALAT1 mRNA in LTK cells (fibroblasts)

The following oligonucleotides have been generated and tested accordingly:

compound ID number	Sequence of	Calculated mass value	Measured value of quality
				#
24	GAGttacttgcca*AmCT	5321.3	5321.7
				#25	GAGt*tacttgcca*AmCT	5363.3	5363.4
#26	GAGt°tacttgcca°AmCT	5331.3	5331.9
				#27	GAGttacttgcca°AmCT	5305.2	5304.9

PACE phosphorothioate modifications between adjacent nucleotides

PACE phosphodiester modification between adjacent nucleotides

A、G、^mC. T represents LNA nucleotide

a. g, c, t represent DNA nucleotides

All other bonds being prepared as phosphorothioates

The above compounds targeting Malat-1 were tested in mouse fibroblasts (LTK cells) by autonomous uptake for 72 hours at a range of concentrations to determine compound potency (IC 50).

Concentration range of LTK cells: 50 μ M, 1/2 log dilution, 8 concentrations.

RNA levels of malt 1 were quantified using qPCR (normalized to GAPDH levels) and IC50 values were determined.

IC50 results are shown in the above table, indicating that this chemical modification is well tolerated in target knockdown (as exemplified herein for disease-associated skeletal muscle cells).

Example 8: target mRNA levels in the heart were measured with a dose of 15mg/kg (Malat1)

Mice (C57/BL6) were administered a 15mg/kg dose of oligonucleotide (n-5) subcutaneously in three doses on days 1,2 and 3. Mice were sacrificed on day 8 and heart MALAT-1RNA reduction was measured. The parent compound was administered in two doses of 3 x 15mg/kg and 3 x 30 mg/kg.

The results are shown in fig. 4.

The in vivo results show that Thio-PACE-modified compound #24 is approximately twice as potent as the reference compound in knocking down MALAT-1 in the heart (efficacy at 15mg/kg is the same as the reference compound at a dose of 30 mg/kg). Compound #25, which has an additional thio-PACE modification introduced at position 12, shows lower efficacy than #24 but still better than the reference compound. The corresponding oxo-PACE analog (#26) showed significantly reduced activity.

The major effect on efficacy with the use of single stranded antisense oligonucleotides according to the invention has been observed in vivo. It should be noted that the dose of the oligonucleotide according to the invention is only 50% of the reference dose.

Example 9: MOE PACE monomer synthesis

9.1.1-cyano-2-methylpropan-2-yl 2-bromoacetate

A solution of 2-bromoacetyl bromide (14.7g, 6.31mL, 72.6mmol, eq: 1.2) was added to a 250mL round bottom flask charged with toluene (67.2 mL). 3-hydroxy-3-methylbutyronitrile (6g, 6.28ml, 60.5mmol, eq: 1) was added slowly with stirring. The round bottom flask was fitted with a Friedrich condenser and a drying tube to an acid separator (containing aqueous NaOH). The reaction mixture was heated to reflux and refluxed overnight. The reaction was allowed to cool to room temperature and the compound was then concentrated in vacuo to give an oil. The crude oil was purified by Combiflash chromatography using ethyl acetate/hexane as gradient, eluting the product in 30% ethyl acetate in hexane to give 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (8.14g, 37mmol, 58% yield).¹H NMR(CHLOROFORM-d,300MHz)δ3.8(s,2H),2.9(s,2H),1.6(s,6H)。

9.2.1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate

Anhydrous THF (69.4mL), 1-chloro-N, N' -tetraisopropyl phosphine diamine (7.75g, 29mmol, eq: 1) and a magnetic stir bar were added to a 250mL round bottom flask, the flask was stoppered, and the solution was stirred until the phosphine was dissolved. After dissolution, anhydrous diethyl ether (41.6ml) was added. 1-cyano-2-methylpropan-2-yl 2-bromoacetate (7.03g, 32mmol, equivalent: 1.1) was placed in a 100mL round-bottomed flask, and anhydrous THF (34.7mL) was added. Zinc (2.85g, 43.6mmol, eq: 1.5), anhydrous diethyl ether (22.2mL) and a magnetic stir bar were placed in a 500mL three-necked round bottom flask fitted with a Friedrich condenser. Phosphine (36mL) and bromoacetate solution (1)0mL) was added to a three-neck round-bottom flask. The reaction mixture was then heated at reflux until an exothermic reaction was evident (slightly cloudy, colorless reaction became clear and light yellow). The reaction was continued at reflux by adding the remaining phosphine and bromoacetate solution. Once the addition was complete, the reaction was held at reflux for 45 minutes by heating, allowed to cool to room temperature and passed³¹P NMR analysis was complete. The starting material at δ -135 ppm was converted to a single product at δ -48 ppm. The cooled reaction mixture was concentrated in vacuo to a viscous oil. The resulting viscous oil was dissolved with anhydrous heptane. The solid formed was then dissolved in acetonitrile and the solution was extracted twice with anhydrous heptane. By passing³¹P NMR analysis of the acetonitrile solution determined that no product was present at δ 48ppm and was discarded. All heptane fractions (top layer) were combined and concentrated in vacuo to give a light yellow oil. It was then dried under high vacuum overnight. After drying overnight, the product was obtained as a beautiful white solid (7.096g, 19mmol, 62% yield).¹H NMR(CHLOROFORM-d,300MHz)δ3.3-3.5(m,4H),2.9(s,2H),2.7(d,2H),1.60(s,6H),1.3(m,24H)。

(1-cyano-2-methylpropan-2-yl) 2- [ [ di (propan-2-yl) amino ] - [ rac- (2R,5R) -2- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -4- (2-methoxyethoxy) -5- (5-methyl-2, 4-dioxopyrimidin-1-yl) oxocyclopentan-3-yl ] oxyphosphonyl ] acetate

Mixing 5-methyl-1- [ rac- (2R,5R) -4-hydroxy-3- (2-methoxyethoxy) -5- [ [ rac- (2E) -1, 1-bis (4-methoxyphenyl) -2- [ rac- (Z) -prop-1-enyl]Penta-2, 4-dienyloxy]Methyl radical]Oxocyclopentan-2-yl radical]Pyrimidine-2, 4-dione (800mg, 1.29mmol, eq: 1) was dissolved in anhydrous DCM (16.2ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (721mg, 1.94mmol, eq: 1.5) was added to the reaction mixture. After the reaction components were completely dissolved, 4,5-DCI (122mg, 1.03mmol, equivalent: 0.8) was added to the reaction mixture. The reaction mixture was then stirred under argon at room temperature overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate) analyzed the extent of reaction. By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. After completion, the reaction was quenched by addition of triethylamine (105mg, 144. mu.l, 1.03mmol, eq.: 0.8). After 5 minutes, the reaction mixture was concentrated in vacuo to a viscous oil using a rotary evaporator. The viscous oil was redissolved in a minimum volume of ethyl acetate and added to the top of a silica gel column pre-equilibrated with 80/20 ethyl acetate/heptane to collect the product. The product containing fractions were combined and concentrated in vacuo on a rotary evaporator to a foam, which was redissolved in a minimum volume of anhydrous DCM and added dropwise to rapidly stirred anhydrous heptane. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 736mg of the title compound as a white solid (736mg, 61% yield). LCMS (ES +) found: 889.5 g/mol.

(1-cyano-2-methylpropan-2-yl) 2- [ [ bis (prop-2-yl) amino ] - [ rac- (2R,5R) -5- (6-benzamidopurin-9-yl) -2- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -4- (2-methoxyethoxy) oxocyclopentan-3-yl ] oxyphosphonyl ] acetate

Rac-N- (9- ((2R,5R) -5- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4-hydroxy-3- (2-methoxyethoxy) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (600mg, 0.82mmol, eq: 1) was dissolved in anhydrous DCM (10.2ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (457mg, 1.23mmol, eq: 1.5) was added to the reaction mixture. After the reaction components were completely dissolved, 4,5-DCI (77.5mg, 0.66mmol, equivalent: 0.8) was added to the reaction mixture. The reaction mixture was then stirred under argon at room temperature overnight and passed³¹PNMR and silica TLC (elution with ethyl acetate) analysis of the reactionDegree of the disease. By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. After completion, the reaction was quenched by addition of triethylamine (66.4mg, 91.4. mu.l, 0.65mmol, eq.: 0.8). After 5 minutes, the reaction mixture was concentrated in vacuo to a viscous oil using a rotary evaporator. The viscous oil was redissolved in a minimum volume of ethyl acetate and added to the top of a silica gel column pre-equilibrated with 80/20 ethyl acetate/heptane to collect the product. The product containing fractions were combined and concentrated in vacuo on a rotary evaporator to a foam, which was redissolved in a minimum volume of anhydrous DCM and added dropwise to rapidly stirred anhydrous heptane. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 260mg of the title compound as a white solid (260mg, 32% yield). LCMS (ES +) found: 1002.5 g/mol.

(1-cyano-2-methylpropan-2-yl) 2- [ [ bis (propan-2-yl) amino ] - [ rac- (2R,5R) -2- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -4- (2-methoxyethoxy) -5- [2- (2-methylpropanamino) -6-oxo-1H-purin-9-yl ] oxocyclopentan-3-yl ] oxyphosphonyl ] acetate

2-methyl-N- [ 6-oxo-9- [ rac- (2R,5R) -5- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] is]Methyl radical]-4-hydroxy-3- (2-methoxyethoxy) oxocyclopentan-2-yl]-1H-purin-2-yl]Propionamide (700mg, 0.98mmol, eq: 1) was dissolved in anhydrous DCM (12.3ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (546mg, 1.47mmol, eq: 1.5) was added to the reaction mixture. After the reaction components were completely dissolved, 4,5-DCI (93mg, 0.79mmol, equivalent: 0.8) was added to the reaction mixture. The reaction mixture was then stirred under argon at room temperature overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate) analyzed the extent of reaction. By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. After completion, the reaction was quenched by addition of triethylamine (80mg, 109. mu.l, 0.79mmol, eq.: 0.8). After 5 minutes, the reaction mixture was concentrated in vacuo to a viscous oil using a rotary evaporator. The viscous oil was redissolved in a minimum volume of ethyl acetate and added to the top of a silica gel column pre-equilibrated with ethyl acetate to collect the product. The product containing fractions were combined and concentrated in vacuo on a rotary evaporator to a foam, which was redissolved in a minimum volume of anhydrous DCM and added dropwise to rapidly stirred anhydrous heptane. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 520mg of the title compound as a white solid (520mg, 49% yield). LCMS (ES +) found: 984.5 g/mol.

(1-cyano-2-methylpropan-2-yl) 2- [ [ di (propan-2-yl) amino ] - [ rac- (2R,5R) -5- (4-benzamido-5-methyl-2-oxopyrimidin-1-yl) -2- [ [ bis (4-methoxyphenyl) -phenylmethoxy ] methyl ] -4- (2-methoxyethoxy) oxocyclopentan-3-yl ] oxyphosphonyl ] acetate

Reacting N- [ 5-methyl-2-oxo-1- [ rac- (2R,5R) -5- [ [ bis (4-methoxyphenyl) -phenylmethoxy group]Methyl radical]-4-hydroxy-3- (2-methoxyethoxy) oxocyclopentan-2-yl]Pyrimidin-4-yl]Benzamide (950mg, 1.32mmol, eq: 1) was dissolved in anhydrous DCM (16.5ml) and 1-cyano-2-methylpropan-2-yl 2- (bis (diisopropylamino) phosphono) acetate (733mg, 1.97mmol, eq: 1.5) was added to the reaction mixture. After the reaction components were completely dissolved, 4,5-DCI (124mg, 1.05mmol, equivalent: 0.8) was added to the reaction mixture. The reaction mixture was then stirred under argon at room temperature overnight and passed³¹P NMR and silica gel TLC (eluted with ethyl acetate) analyzed the extent of reaction. By point-by-point conversion on TLC to the faster eluting product and by phosphinodiamine acetate³¹Complete loss of P NMR signal confirms reaction completion. After completion, the reaction mixture was purified by addition of triethylamine (107mg, 147. mu.l, 1.05mmol,equivalent weight: 0.8) quenching the reaction. After 5 minutes, the reaction mixture was concentrated in vacuo to a viscous oil using a rotary evaporator. The viscous oil was redissolved in a minimum volume of ethyl acetate and added to the top of a silica gel column pre-equilibrated with 80/20 ethyl acetate/heptane to collect the product. The product containing fractions were combined and concentrated in vacuo on a rotary evaporator to a foam, which was redissolved in a minimum volume of anhydrous DCM and added dropwise to rapidly stirred anhydrous heptane. The solid precipitate was isolated by filtration and dried under vacuum overnight to give 722mg of the title compound as a pale yellow solid (722mg, 55% yield). LCMS (ES +) found: 992.4 g/mol.

Example 10: oligonucleotide synthesis

Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer from Bioautomation. Using controlled aperture glass supports with universal joints

The synthesis was performed on a 1. mu. mol scale.

In a standard cycling procedure for coupling standard DNA and LNA phosphoramidites, 3% (w/v) of CH in dichloroacetic acid was used₂Cl₂The solution was DMT deprotected in three applications, 230. mu.L each, for 105 seconds. 95 μ L of a 0.1M acetonitrile solution (or for LNA-^MeC building a block of acetonitrile/CH₂Cl₂1:1 solution) and 110. mu.L of 0.25M 5- [3, 5-bis (trifluoromethyl) phenyl]-2H-tetrazole as activator and a coupling time of 180 seconds was coupled three times with the corresponding phosphoramidite. Sulfurization was carried out in one application using a 0.1M acetonitrile/pyridine solution of 3-amino-1, 2, 4-dithiazole-5-thione, at a volume of 200. mu.L, for 3 minutes. Use 0.02M I₂THF/pyr/H of₂The solution of O88/10/2 was oxidized in one application for 3 minutes. Using THF/lutidine/Ac₂O8: 1:1(CapA, 75. mu. mol) and THF/N-methylimidazole 8:2(CapB, 75. mu. mol) were capped for 70 seconds.

The synthesis cycle for the introduction of MOE PACE included the use of 3% (w/v) of the CH of dichloroacetic acid₂Cl₂DMT deprotection of the solution was performed in three applications, 23 each0 μ L for 105 seconds. With 95. mu.L of a 0.1M acetonitrile solution and 110. mu.L of a 0.25M 5- [3, 5-bis (trifluoromethyl) phenyl group]The freshly prepared MOE PACE phosphoramidite was coupled twice with-2H-tetrazole solution as the activator and a coupling time of 15 minutes. Sulfurization was carried out using 0.1M 3-amino-1, 2, 4-dithiazole-5-thione in acetonitrile/pyridine solution in one application for 3 minutes. Use 0.02M I₂THF/pyr/H of₂The solution of O88/10/2 was oxidized in one application for 3 minutes. Using THF/lutidine/Ac₂O8: 1:1(CapA, 75. mu. mol) and THF/N-methylimidazole 8:2(CapB, 75. mu. mol) were capped for 70 seconds.

After synthesis, 1.5% DBU was in anhydrous CH₃The solution in CN was carefully passed through the column several times to deprotect the dimethylcyanoethyl protecting group and prevent alkylation of the base during deprotection. Then allowed to stand at room temperature for 60 minutes. The solution was then discarded and 2-3mL of anhydrous CH was used₃CN washes the column. It was then dried under a stream of argon. The CPG was then carefully transferred to a 4mL vial to which was added 1mL of 40% MeNH₂And stirred at 55 ℃ for 15 minutes.

Crude DMT-on oligonucleotides were purified by RP-HPLC purification using a C18 column followed by DMT removal by precipitation with 80% aqueous acetic acid and ethanol or by column purification. MOE PACE phosphoramidites are synthesized in basel. Normal phosphoramidite and all reagents for solid phase synthesis were ordered from Sigma Aldrich.

Example 11: in vitro potency and efficacy of oligonucleotides targeting MALAT1 mRNA in human HeLa cells at different concentrations of the dose-response curve

HeLa cell lines were purchased from ATCC and maintained at 37 ℃ and 5% CO according to supplier's recommendations₂In a humidified incubator. For the assay, cells were seeded at 3000 cells/well in medium in 96-well plates. Cells were cultured for 24 hours, and then oligonucleotides dissolved in PBS were added. Concentration range of oligonucleotide: the maximum concentration is 25. mu.M, and 1:1 dilution is carried out in 8 steps. Three days after addition of the oligonucleotides, cells were harvested. RNA was extracted using the PureLink Pro 96RNA purification kit (Thermo Fisher Scientific) according to the manufacturer's instructions,and eluted in 50. mu.l of water. The RNA was then diluted 10-fold with DNase/RNase free water (Gibco) and heated to 90 ℃ for one minute.

For gene expression analysis, qScript was used^TMXLT One-Step RT-qPCR

Low ROX^TM(Quanntadio) one-step RT-qPCR was performed in a duplex setting. The following TaqMan primer assays were used for qPCR: MALAT1, Hs00273907_ s1(FAM-MGB) and the endogenous control GAPDH. All primer sets were purchased from Thermo Fisher Scientific. Relative expression levels of MALAT1 mRNA were shown as a percentage of control (PBS-treated cells), and IC has been determined using GraphPad Prism7 from data for n-2 biological replicates₅₀The value is obtained.

The results are provided in the table.

Bold letters t, a, g, c represent MOE modifications.

(ps) phosphorothioate modifications between adjacent nucleotides

(po) phosphodiester modifications between adjacent nucleotides

PACE phosphorothioate modifications between adjacent nucleotides

PACE phosphodiester modification between adjacent nucleotides

A、G、^mC. T represents LNA nucleotide

a. g, c, t represent DNA nucleotides

All other bonds were prepared as phosphorothioates.

Claims (17)

1. A compound of formula (I-a) or a pharmaceutically acceptable salt thereof

Wherein

R²Is alkoxy, alkoxyalkoxy or amino; and is

R⁴Is hydrogen; or

R⁴And R²Together form X-Y;

x is oxygen, sulfur or-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(＝CR^aR^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-；-O-NR^a-、-NR^a-O-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

Y is oxygen, sulfur, - (CR)^aR^b)_n-、-CR^aR^b-O-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

With the proviso that-X-Y-is not-O-O-, Si (R)^a)₂-Si(R^a)₂-、-SO₂-SO₂-、-C(R^a)＝C(R^b)-C(R^a)＝C(R^b)、-C(R^a)＝N-C(R^a)＝N-、-C(R^a)＝N-C(R^a)＝C(R^b)、-C(R^a)＝C(R^b)-C(R^a) N-or-Se-;

j is oxygen, sulfur, ═ CH₂Or ═ N (R)^a)；

R^aAnd R^bIndependently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, mercapto, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, ureido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, mercaptoalkylthio, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC (═ X^a)R^c、-OC(＝X^a)NR^cR^dand-NR^eC(＝X^a)NR^cR^d；

Or two geminal R^aAnd R^bTogether form an optionally substituted methylene group;

or two geminal R^aAnd R^bTogether with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl group, and only one carbon atom in-X-Y-has this condition;

wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy, and substituted methylene are alkyl, alkenyl, alkynyl, and methylene substituted with 1 to 3 substituents independently selected from the group consisting of: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl and heteroaryl;

X^ais oxygen, sulfur or-NR^c；

R^c、R^dAnd R^eIndependently selected from hydrogen and alkyl;

R⁵is a hydroxyl protecting group;

R^xis cyanoalkyl or alkyl;

R^yis dialkylamino or pyrrolidinyl;

nu is a nucleobase or a protected nucleobase; and is

n is 1,2 or 3.

2. The compound of claim 1, which is of formula (II) or a pharmaceutically acceptable salt thereof

Wherein

X is oxygen, sulfur or-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(＝CR^aR^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-；-O-NR^a-、-NR^a-O-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

Y is oxygen, sulfur, - (CR)^aR^b)_n-、-CR^aR^b-O-CR^aR^b-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-Si(R^a)₂-、-SO₂-、-NR^a-、-C(＝J)-、Se、-O-NR^a-、-NR^a-CR^aR^b-、-N(R^a) -O-or-O-CR^aR^b-；

With the proviso that-X-Y-is not-O-O-, Si (R)^a)₂-Si(R^a)₂-、-SO₂-SO₂-、-C(R^a)＝C(R^b)-C(R^a)＝C(R^b)、-C(R^a)＝N-C(R^a)＝N-、-C(R^a)＝N-C(R^a)＝C(R^b)、-C(R^a)＝C(R^b)-C(R^a) N-or-Se-;

j is oxygen, sulfur, ═ CH₂Or ═ N (R)^a)；

R^aAnd R^bIndependently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, mercapto, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, ureido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, mercaptoalkylthio, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC (═ X^a)R^c、-OC(＝X^a)NR^cR^dand-NR^eC(＝X^a)NR^cR^d；

Or two geminal R^aAnd R^bTogether form an optionally substituted methylene group;

or two geminal R^aAnd R^bTogether with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl group, and only one carbon atom in-X-Y-has this condition;

wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy, and substituted methylene are alkyl, alkenyl, alkynyl, and methylene substituted with 1 to 3 substituents independently selected from the group consisting of: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl and heteroaryl;

X^ais oxygen, sulfur or-NR^c；

R^c、R^dAnd R^eIndependently selected from hydrogen and alkyl;

R⁵is a hydroxyl protecting group;

R^xis cyanoalkyl or alkyl;

R^yis dialkylamino or pyrrolidinyl;

nu is a nucleobase or a protected nucleobase; and is

n is 1,2 or 3.

3. The compound of claim 1, having formula (VI)

Wherein R is²、R⁵、R^x、R^yAnd Nu is as defined in claim 1.

4. A compound according to claim 1 or 2, wherein-X-Y-is-CH₂-O-、-CH(CH₃) -O-or-CH₂CH₂-O-。

5. The compound of any one of claims 1 to 4, having formula (III), (IV) or (VII)

Wherein R is⁵、R^x、R^yAnd Nu is as defined in claim 1.

6. A compound according to any one of claims 1 to 5, wherein R^xIs 2-cyano-1, 1-dimethyl-ethyl.

7. A compound according to any one of claims 1 to 6, wherein R^yIs diisopropylamino or pyrrolidinyl.

8. A compound according to any one of claims 1 to 7, wherein R^yIs a dialkylamino group.

9. A compound according to any one of claims 1 to 8, wherein R^yIs diisopropylamino.

10. The compound of any one of claims 1 to 8, having formula (V) or (VIII)

Wherein R is⁵And Nu is as defined in claim 1.

11. The compound according to any one of claims 1 to 10, wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil or protected uracil.

12. A compound according to any one of claims 1 to 11 selected from

And

13. a process for the manufacture of a compound of formula (I-a) according to any one of claims 1 to 12, comprising reacting a compound of formula (E)

And formula P (R)^y)₂(CH₂)COO(R^x) In the presence of a coupling agent, wherein X, Y, R⁵、Nu、R^xAnd R^yAs defined in any one of claims 1 to 12.

14. The method of claim 13, comprising reacting a compound of formula (C) or (D)

And formula P (R)^y)₂(CH₂)COO(R^x) In the presence of a coupling agent, wherein X, Y, R⁵、Nu、R^xAnd R^yAs defined in any one of claims 1 to 12.

15. The method of claim 13 or 14, wherein the coupling agent is 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, or 4, 5-Dicyanoimidazole (DCI).

16. Use of a compound according to any one of claims 1 to 12 in the manufacture of an oligonucleotide.

17. The invention as hereinbefore described.

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